Methods, reagents and cells for biosynthesizing compounds

ABSTRACT

This document describes biochemical pathways for producing 5-hydroxypentanoate methyl ester and pentanoic acid pentyl ester using one or more of a fatty acid O-methyltransferase, an alcohol O-acetyltransferase, and a monooxygenase, as well as recombinant hosts expressing one or more of such exogenous enzymes. 5-hydroxypentanoate methyl esters and pentanoic acid pentyl esters can be enzymatically converted to glutaric acid, 5-aminopentanoate, 5-hydroxypentanoate, cadaverine, or 1,5-pentanediol.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.Nos. 62/012,659, filed Jun. 16, 2014, 62/012,666, filed Jun. 16, 2014,and 62/012,604, filed Jun. 16, 2014, the disclosures of each of whichare incorporated herein by reference in their entirety.

TECHNICAL FIELD

This invention relates to methods for biosynthesizing5-hydroxypentanoate methyl ester and pentanoic acid pentyl ester usingone or more isolated enzymes such as a fatty acid O-methyltransferase,an alcohol O-acetyltransferase, and a monooxygenase, and to recombinanthost cells expressing one or more such enzymes. This invention alsorelates to methods for enzymatically converting 5-hydroxypentanoatemethyl ester and pentanoic acid pentyl ester to 5-hydroxypentanoate and1,5-pentanediol using one or more enzymes such as an a monooxygenase, ademethylase, or an esterase, and recombinant hosts expressing one ormore such enzymes. In addition, this invention relates to enzymaticallyconverting 5-hydroxypentanoate and/or 1,5-pentanediol to glutaric acid,5-aminopentanoic acid, cadaverine or 1,5-pentanediol (hereafter “C5building blocks) and recombinant hosts producing such C5 buildingblocks.

BACKGROUND

Nylons are polyamides which are generally synthesized by thecondensation polymerization of a diamine with a dicarboxylic acid.Similarly, Nylons may be produced by the condensation polymerization oflactams. A ubiquitous nylon is Nylon 6,6, which is produced bycondensation polymerization of hexamethylenediamine (HMD) and adipicacid. Nylon 6 can be produced by a ring opening polymerization ofcaprolactam (Anton & Baird, Polyamides Fibers, Encyclopedia of PolymerScience and Technology, 2001).

Nylon 5 and Nylon 5,5 represent novel polyamides with value-addedcharacteristics compared to Nylon 6 and Nylon 6,6. Nylon 5 is producedby polymerisation of 5-aminopentanoic acid, whereas Nylon 5,5 isproduced by condensation polymerisation of glutaric acid and cadaverine.No economically viable petrochemical routes exist to producing themonomers for Nylon 5 and Nylon 5,5.

Given no economically viable petrochemical monomer feedstocks,biotechnology offers an alternative approach via biocatalysis.Biocatalysis is the use of biological catalysts, such as enzymes, toperform biochemical transformations of organic compounds.

Both bioderived feedstocks and petrochemical feedstocks are viablestarting materials for the biocatalysis processes.

Accordingly, against this background, it is clear that there is a needfor sustainable methods for producing one or more of glutaric acid,5-hydroxypentanoate, 5-aminopentanoate, cadaverine and 1,5-pentanediol(hereafter “C5 building blocks”) wherein the methods are biocatalystbased.

However, no wild-type prokaryote or eukaryote naturally overproduces orexcretes such C5 building blocks to the extracellular environment.Nevertheless, the metabolism of glutaric acid has been reported.

The dicarboxylic acid glutaric acid is converted efficiently as a carbonsource by a number of bacteria and yeasts via β-oxidation into centralmetabolites. Decarboxylation of Coenzyme A (CoA) activated glutarate tocrotonyl-CoA facilitates further catabolism via β-oxidation.

The optimality principle states that microorganisms regulate theirbiochemical networks to support maximum biomass growth. Beyond the needfor expressing heterologous pathways in a host organism, directingcarbon flux towards C5 building blocks that serve as carbon sourcesrather than as biomass growth constituents, contradicts the optimalityprinciple. For example, transferring the 1-butanol pathway fromClostridium species into other production strains has often fallen shortby an order of magnitude compared to the production performance ofnative producers (Shen et al., Appl. Environ. Microbiol., 2011,77(9):2905-2915).

The efficient synthesis of the seven carbon aliphatic backbone precursoris a key consideration in synthesizing one or more C5 building blocksprior to forming terminal functional groups, such as carboxyl, amine orhydroxyl groups, on the C5 aliphatic backbone.

SUMMARY

This document is based at least in part on the discovery that it ispossible to construct biochemical pathways for producing a seven carbonchain aliphatic backbone precursor in which one or two functionalgroups, i.e., carboxyl, amine, or hydroxyl, can be formed, leading tothe synthesis of one or more of glutaric acid, 5-aminopentanoate,5-hydroxypentanoate, cadaverine, and 1,5-pentanediol (hereafter “C5building blocks). Glutaric acid and glutarate, 5-hydroxypentanoic acidand 5-hydroxypentanoate, and 5-aminopentanoic and 5-aminopentanoate areused interchangeably herein to refer to the relevant compound in any ofits neutral or ionized forms, including any salt forms thereof. It isunderstood by those skilled in the art that the specific form willdepend on pH.

One of skill in the art understands that compounds containing carboxylicacid groups (including, but not limited to, organic monoacids,hydroxyacids, aminoacids, and dicarboxylic acids) are formed orconverted to their ionic salt form when an acidic proton present in theparent compound either is replaced by a metal ion, e.g., an alkali metalion, an alkaline earth ion, or an aluminum ion; or coordinates with anorganic base. Acceptable organic bases include, but are not limited to,ethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like. Acceptable inorganic bases include, butare not limited to, aluminum hydroxide, calcium hydroxide, potassiumhydroxide, sodium carbonate, sodium hydroxide, and the like. A salt ofthe present invention is isolated as a salt or converted to the freeacid by reducing the pH to below the pKa, through addition of acid ortreatment with an acidic ion exchange resin.

One of skill in the art understands that compounds containing aminegroups (including, but not limited to, organic amines, aminoacids, anddiamines) are formed or converted to their ionic salt form, for example,by addition of an acidic proton to the amine to form the ammonium salt,formed with inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, and the like; or formedwith organic acids including, but not limited to, acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like. Acceptable inorganicbases include, but are not limited to, aluminum hydroxide, calciumhydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, andthe like. A salt of the present invention is isolated as a salt orconverted to the free amine by raising the pH to above the pKb throughaddition of base or treatment with a basic ion exchange resin.

One of skill in the art understands that compounds containing both aminegroups and carboxylic acid groups (including, but not limited to,aminoacids) are formed or converted to their ionic salt form byeither 1) acid addition salts, formed with inorganic acids including,but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid,nitric acid, phosphoric acid, and the like; or formed with organic acidsincluding, but not limited to, acetic acid, propionic acid, hexanoicacid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lacticacid, malonic acid, succinic acid, malic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,2-naphthalenesulfonic acid,4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like. Acceptable inorganicbases include, but are not limited to, aluminum hydroxide, calciumhydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, andthe like, or 2) when an acidic proton present in the parent compoundeither is replaced by a metal ion, e.g., an alkali metal ion, analkaline earth ion, or an aluminum ion; or coordinates with an organicbase. Acceptable organic bases include, but are not limited to,ethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like. Acceptable inorganic bases include, butare not limited to, aluminum hydroxide, calcium hydroxide, potassiumhydroxide, sodium carbonate, sodium hydroxide, and the like. A salt canof the present invention is isolated as a salt or converted to the freeacid by reducing the pH to below the pKa through addition of acid ortreatment with an acidic ion exchange resin.

Pathways, metabolic engineering and cultivation strategies describedherein can rely on producing pentanoate methyl ester from pentanoateusing, for example, a fatty acid O-methyltransferase and producing5-hydroxypentanoate methyl ester from pentanoate methyl ester using, forexample, a monooxygenase. 5-hydroxypentanoate can be produced from5-hydroxypentanoate methyl ester using, for example, a demethylase or anesterase.

Pathways, metabolic engineering and cultivation strategies describedherein also can rely on producing pentanoic acid pentyl ester using, forexample, an alcohol O-acetyltransferase and producing 5-hydroxypentanoicacid pentyl ester, 5-hydroxypentanoic acid 5-hydroxypentyl ester and/orpentanoic acid 5-hydroxypentyl ester from pentanoic acid pentyl esterusing, for example, a monooxygenase. 5-hydroxypentanoate can be producedfrom 5-hydroxypentanoic acid pentyl ester and/or 5-hydroxypentanoic acid5-hydroxypentyl ester using, for example, an esterase. 1,5-pentanediolcan be produced from pentanoic acid 5-hydroxypentyl ester and/or5-hydroxypentanoic acid 5-hydroxypentyl ester using, for example, anesterase.

CoA-dependent elongation enzymes or homologs associated with the carbonstorage pathways from polyhydroxyalkanoate accumulating bacteria areuseful for producing precursor molecules. See, e.g., FIGS. 1-2.

In the face of the optimality principle, the inventors discoveredsurprisingly that appropriate non-natural pathways, feedstocks, hostmicroorganisms, attenuation strategies to the host's biochemical networkand cultivation strategies may be combined to efficiently produce one ormore C5 building blocks.

In some embodiments, the C5 aliphatic backbone for conversion to a C5building block can be formed from acetyl-CoA and propanoyl-CoA via twocycles of CoA-dependent carbon chain elongation using either NADH orNADPH dependent enzymes. See FIG. 1 and FIG. 2.

In some embodiments, an enzyme in the CoA-dependent carbon chainelongation pathway generating the C5 aliphatic backbone purposefullycontains irreversible enzymatic steps.

In some embodiments, the terminal carboxyl groups can be enzymaticallyformed using a thioesterase, an aldehyde dehydrogenase, a7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a5-oxopentanoate dehydrogenase or a monooxygenase. See FIG. 3 and FIG. 4.

In some embodiments, the terminal amine groups can be enzymaticallyformed using a ω-transaminase or a deacetylase. See FIG. 5 and FIG. 6.

In some embodiments, the terminal hydroxyl group can be enzymaticallyformed using a monooxygenase, an esterase, or an alcohol dehydrogenase.See FIG. 3, FIG. 7 and FIG. 8. A monooxygenase (e.g., in combinationwith an oxidoreductase and/or ferredoxin) or an alcohol dehydrogenasecan enzymatically form a hydroxyl group. The monooxygenase can have atleast 70% sequence identity to any one of the amino acid sequences setforth in SEQ ID NOs: 14-16 or 28-29. An esterase can have at least 70%identity to the amino acid sequence set forth in SEQ ID NO: 27.

A ω-transaminase or a deacetylase can enzymatically form an amine group.The ω-transaminase can have at least 70% sequence identity to any one ofthe amino acid sequences set forth in SEQ ID NOs. 8-13.

A thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoatedehydrogenase, a 5-oxopentanoate dehydrogenase, or a 6-oxohexanoatedehydrogenase can enzymatically form a terminal carboxyl group. Thethioesterase can have at least 70% sequence identity to the amino acidsequence set forth in SEQ ID NO: 1, SEQ ID NO: 33, and/or SEQ ID NO: 34.

A carboxylate reductase (e.g., in combination with a phosphopantetheinyltransferase) can form a terminal aldehyde group as an intermediate informing the product. The carboxylate reductase can have at least 70%sequence identity to any one of the amino acid sequences set forth inSEQ ID NOs: 2-7.

Any of the methods can be performed in a recombinant host byfermentation. The host can be subjected to a cultivation strategy underaerobic, anaerobic, or micro-aerobic cultivation conditions. The hostcan be cultured under conditions of nutrient limitation such asphosphate, oxygen or nitrogen limitation. The host can be retained usinga ceramic membrane to maintain a high cell density during fermentation.

In any of the methods, the host's tolerance to high concentrations of aC5 building block can be improved through continuous cultivation in aselective environment.

The principal carbon source fed to the fermentation can derive frombiological or non-biological feedstocks. In some embodiments, thebiological feedstock is, includes, or derives from, monosaccharides,disaccharides, lignocellulose, hemicellulose, cellulose, lignin,levulinic acid and formic acid, triglycerides, glycerol, fatty acids,agricultural waste, condensed distillers' solubles, or municipal waste.

In some embodiments, the non-biological feedstock is or derives fromnatural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate, non-volatileresidue (NVR) or a caustic wash waste stream from cyclohexane oxidationprocesses, or a terephthalic acid/isophthalic acid mixture waste stream.

This document also features a recombinant host that includes at leastone exogenous nucleic acid encoding a fatty acid O-methyltransferase anda monooxygenase, and produce 5-hydroxypentanoate methyl ester. Such ahost further can include a demethylase or esterase and further produce5-hydroxypentanoate. Such hosts further can include (i) a β-ketothiolaseor an acetyl-CoA carboxylase and a β-ketoacyl-[acp] synthase, (ii) a3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-CoA reductase, (iii) anenoyl-CoA hydratase, and (iv) a trans-2-enoyl-CoA reductase. The hostsalso further can include one or more of a thioesterase, an aldehydedehydrogenase, or a butanal dehydrogenase.

This document also features a recombinant host that includes at leastone exogenous nucleic acid encoding an alcohol O-acetyltransferase andproduce pentanoic acid pentyl ester. Such a host further can include amonooxygenase and an esterase and further produce 5-hydroxypentanoateand/or 1,5-pentanediol. Such hosts further can include (i) aβ-ketothiolase or an acetyl-CoA carboxylase and a β-ketoacyl-[acp]synthase, (ii) a 3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl-CoAreductase, (iii) an enoyl-CoA hydratase, and (iv) a trans-2-enoyl-CoAreductase. The hosts also further can include one or more of athioesterase, a carboxylate reductase, an aldehyde dehydrogenase, abutanal or acetaldehyde dehydrogenase, or an alcohol dehydrogenase.

A recombinant host producing 5-hydroxypentanoate further can include oneor more of a monooxygenase, an alcohol dehydrogenase, an aldehydedehydrogenase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoatedehydrogenase, a 4-hydroxybutyrate dehydrogenase, a 7-oxoheptanoatedehydrogenase, a 6-oxohexanoate dehydrogenase, or a 5-oxopentanoatedehydrogenase, the host further producing glutaric acid or glutaratesemialdehyde.

A recombinant host producing 5-hydroxypentanoate further can include oneor more of a monooxygenase, a transaminase, a 6-hydroxyhexanoatedehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyratedehydrogenase, and an alcohol dehydrogenase, wherein the host furtherproduces 5-aminopentanoate.

A recombinant host producing 5-hydroxypentanoate or 5-aminopentanoatefurther can include one or more of a carboxylate reductase, aω-transaminase, a deacetylase, a N-acetyl transferase, or an alcoholdehydrogenase, the host further producing cadaverine.

A recombinant host producing 5-hydroxypentanoate further can include acarboxylate reductase or an alcohol dehydrogenase, wherein the hostfurther produces 1,5-pentanediol.

The recombinant host can be a prokaryote, e.g., from the genusEscherichia such as Escherichia coli; from the genus Clostridia such asClostridium ljungdahlii, Clostridium autoethanogenum or Clostridiumkluyveri; from the genus Corynebacteria such as Corynebacteriumglutamicum; from the genus Cupriavidus such as Cupriavidus necator orCupriavidus metallidurans; from the genus Pseudomonas such asPseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans;from the genus Delftia acidovorans, from the genus Bacillus such asBacillus subtillis; from the genes Lactobacillus such as Lactobacillusdelbrueckii; from the genus Lactococcus such as Lactococcus lactis orfrom the genus Rhodococcus such as Rhodococcus equi.

The recombinant host can be a eukaryote, e.g., a eukaryote from thegenus Aspergillus such as Aspergillus niger; from the genusSaccharomyces such as Saccharomyces cerevisiae; from the genus Pichiasuch as Pichia pastoris; from the genus Yarrowia such as Yarrowialipolytica, from the genus Issatchenkia such as Issathenkia orientalis,from the genus Debaryomyces such as Debaryomyces hansenii, from thegenus Arxula such as Arxula adenoinivorans, or from the genusKluyveromyces such as Kluyveromyces lactis.

In some embodiments, the host's endogenous biochemical network isattenuated or augmented to (1) ensure the intracellular availability ofacetyl-CoA and propanoyl-CoA, (2) create a cofactor, i.e. NADH or NADPH,imbalance that may be balanced via the formation of a C5 building block,(3) prevent degradation of central metabolites, central precursorsleading to and including C5 building blocks and (4) ensure efficientefflux from the cell.

Any of the recombinant hosts described herein further can include one ormore of the following attenuated enzymes: polyhydroxyalkanoate synthase,an acetyl-CoA thioesterase, a propanoyl-CoA thioesterase, amethylcitrate synthase, an acetyl-CoA specific β-ketothiolase, aphosphotransacetylase forming acetate, an acetate kinase, a lactatedehydrogenase, a menaquinol-fumarate oxidoreductase, a 2-oxoaciddecarboxylase producing isobutanol, an alcohol dehydrogenase formingethanol, a triose phosphate isomerase, a pyruvate decarboxylase, aglucose-6-phosphate isomerase, a transhydrogenase dissipating thecofactor imbalance, a glutamate dehydrogenase specific for the co-factorfor which an imbalance is created, a NADH/NADPH-utilizing glutamatedehydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenaseaccepting C5 building blocks and central precursors as substrates; aglutaryl-CoA dehydrogenase; or a pimeloyl-CoA synthetase.

Any of the recombinant hosts described herein further can overexpressone or more genes encoding: an acetyl-CoA synthetase, a6-phosphogluconate dehydrogenase; a transketolase; a feedback resistantthreonine deaminase; a puridine nucleotide transhydrogenase; a formatedehydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; aglucose-6-phosphate dehydrogenase; a fructose 1,6 diphosphatase; apropionyl-CoA synthetase; a L-alanine dehydrogenase; a L-glutamatedehydrogenase; a L-glutamine synthetase; a lysine transporter; adicarboxylate transporter; and/or a multidrug transporter.

This document also features methods of producing a (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester, the method including enzymaticallyconverting a C₄₋₉ carboxylic acid to a (C₃₋₈ alkyl)-C(═O)OCH₃ ester; andenzymatically converting the (C₃₋₈ alkyl)-C(═O)OCH₃ ester to (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester.

In some embodiments, the C₄₋₉ carboxylic acid can be enzymaticallyconverted to the (C₃₋₉ alkyl)-C(═O)OCH₃ ester using a polypeptide havingfatty acid O-methyltransferase activity. In some embodiments, thepolypeptide having fatty acid O-methyltransferase activity is classifiedunder EC 2.1.1.15. In some embodiments, the polypeptide having fattyacid O-methyltransferase activity can have at least 70% sequenceidentity to an amino acid sequence set forth in SEQ ID NO:23, SEQ IDNO:24, or SEQ ID NO:25.

In some embodiments, the (C₃₋₈ alkyl)-C(═O)OCH₃ ester can beenzymatically converted to the (C₃₋₈ hydroxyalkyl)-C(═O)OCH₃ ester usinga polypeptide having monooxygenase activity. In some embodiments, themonooxygenase is classified under EC 1.14.14.- or EC 1.14.15.-. In someembodiments, the monooxygenase can have at least 70% sequence identityto an amino acid sequence set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO:28 and/or SEQ ID NO:29.

In some embodiments, the C₄₋₉ carboxylic acid can be enzymaticallyproduced from a C₄₋₉ alkanoyl-CoA. In some embodiments, a polypeptidehaving thioesterase activity can enzymatically produce the C₄₋₉carboxylic acid from the C₄₋₉ alkanoyl-CoA. In some embodiments, thethioesterase can have at least 70% sequence identity to the amino acidsequence set forth in SEQ ID NO:1, SEQ ID NO: 33, and/or SEQ ID NO: 34.In some embodiments, a polypeptide having butanal dehydrogenase activityand a polypeptide having aldehyde dehydrogenase activity enzymaticallyproduce the C₄₋₉ carboxylic acid from C₄₋₉ alkanoyl-CoA.

This document also features methods of producing one or morehydroxy-substituted (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) esters. The methodincludes enzymatically converting a C₄₋₉ alkanoyl-CoA to a (C₄₋₉alkyl)-OC(═O)—(C₃₋₈ alkyl) ester; and enzymatically converting the (C₄₋₉alkyl)-OC(═O)—(C₃₋₈ alkyl) ester to any of (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈hydroxyalkyl) ester, (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl)ester, or (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester.

In some embodiments, the C₄₋₉ alkanoyl-CoA can be enzymaticallyconverted to the (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester using apolypeptide having alcohol O-acetyltransferase activity. In someembodiments, the alcohol O-acetyltransferase can have at least 70%sequence identity to the amino acid sequence set forth in SEQ ID NO: 26.

In some embodiments, the (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester can beenzymatically converted to any of (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈hydroxyalkyl) ester, (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl)ester, or (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester using apolypeptide having monooxygenase activity. In some embodiments, thepolypeptide having monooxygenase activity can be classified under EC1.14.14.- or EC 1.14.15.-.

In some embodiments, the method further can include enzymaticallyconverting the (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester or(C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester to a C₄₋₉hydroxyalkanoate. In some embodiments, a polypeptide having esteraseactivity enzymatically converts the (C₄₋₉ hydroxyalkyl)OC(═O)—(C₃₋₈hydroxyalkyl) ester or (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester tothe C₄₋₉ hydroxyalkanoate.

In one aspect, this document features a method for producing abioderived 5-carbon compound. The method for producing a bioderived5-carbon compound can include culturing or growing a recombinant host asdescribed herein under conditions and for a sufficient period of time toproduce the bioderived 5-carbon compound, wherein, optionally, thebioderived 5-carbon compound is selected from the group consisting ofglutaric acid, glutarate semialdehyde, 5-aminopentanoate acid,5-hydroxypentanoate, pentamethylenediamine, 1,5-pentanediol, andcombinations thereof.

In one aspect, this document features composition comprising abioderived 5-carbon compound as described herein and a compound otherthan the bioderived 5-carbon compound, wherein the bioderived 5-carboncompound is selected from the group consisting of glutaric acid,glutarate semialdehyde, 5-aminopentanoate acid, 5-hydroxypentanoate,pentamethylenediamine, 1,5-pentanediol, and combinations thereof. Forexample, the bioderived 5-carbon compound is a cellular portion of ahost cell or an organism.

This document also features a biobased polymer comprising the bioderivedglutaric acid, glutarate semialdehyde, 5-aminopentanoate acid,5-hydroxypentanoate, pentamethylenediamine, 1,5-pentanediol, andcombinations thereof.

This document also features a biobased resin comprising the bioderivedglutaric acid, glutarate semialdehyde, 5-aminopentanoate acid,5-hydroxypentanoate, pentamethylenediamine, 1,5-pentanediol, andcombinations thereof. as well as a molded product obtained by molding abiobased resin.

In another aspect, this document features a process for producing abiobased polymer that includes chemically reacting the bioderivedglutaric acid, glutarate semialdehyde, 5-aminopentanoate acid,5-hydroxypentanoate, pentamethylenediamine, 1,5-pentanediol, andcombinations thereof with itself or another compound in a polymerproducing reaction.

In another aspect, this document features a process for producing abiobased resin that includes chemically reacting the bioderived glutaricacid, glutarate semialdehyde, 5-aminopentanoate acid,5-hydroxypentanoate, pentamethylenediamine, 1,5-pentanediol, andcombinations thereof with itself or another compound in a resinproducing reaction.

In another aspect, this document provides a bio-derived product,bio-based product or fermentation-derived product, wherein said productcomprises:

(i) a composition comprising at least one bio-derived, bio-based orfermentation-derived compound provided herein or in any one of FIGS.1-9, or any combination thereof;

(ii) a bio-derived, bio-based or fermentation-derived polymer comprisingthe bio-derived, bio-based or fermentation-derived composition orcompound of (i), or any combination thereof;

(iii) a bio-derived, bio-based or fermentation-derived resin comprisingthe bio-derived, bio-based or fermentation-derived compound orbio-derived, bio-based or fermentation-derived composition of (i) or anycombination thereof or the bio-derived, bio-based orfermentation-derived polymer of (ii) or any combination thereof;

(iv) a molded substance obtained by molding the bio-derived, bio-basedor fermentation-derived polymer of (ii) or the bio-derived, bio-based orfermentation-derived resin of (iii), or any combination thereof;

(v) a bio-derived, bio-based or fermentation-derived formulationcomprising the bio-derived, bio-based or fermentation-derivedcomposition of (i), bio-derived, bio-based or fermentation-derivedcompound of (i), bio-derived, bio-based or fermentation-derived polymerof (ii), bio-derived, bio-based or fermentation-derived resin of (iii),or bio-derived, bio-based or fermentation-derived molded substance of(iv), or any combination thereof; or

(vi) a bio-derived, bio-based or fermentation-derived semi-solid or anon-semi-solid stream, comprising the bio-derived, bio-based orfermentation-derived composition of (i), bio-derived, bio-based orfermentation-derived compound of (i), bio-derived, bio-based orfermentation-derived polymer of (ii), bio-derived, bio-based orfermentation-derived resin of (iii), bio-derived, bio-based orfermentation-derived formulation of (v), or bio-derived, bio-based orfermentation-derived molded substance of (iv), or any combinationthereof.

Also, described herein is a biochemical network comprising at least oneexogenous nucleic acid encoding a polypeptide having (i) fatty acidO-methyltransferase activity or alcohol O-acetyltransferase activity,(ii) monooxygenase activity, and (iii) esterase or demethylase activity.

Also, described herein is a biochemical network comprising at least oneexogenous nucleic acid encoding a polypeptide having fatty acidO-methyltransferase activity and a polypeptide having monooxygenaseactivity, wherein the biochemical network enzymatically produces5-hydroxypentanoate methyl ester. The biochemical network can furtherinclude a polypeptide having demethylase activity or a polypeptidehaving esterase activity, wherein the polypeptide having demethylaseactivity or a polypeptide having esterase activity enzymatically produce5-hydroxypentanoate.

The biochemical network can further include at least one exogenousnucleic acid encoding a polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity, wherein the polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity enzymatically produce C5 precursor molecules such aspentanoyl-CoA.

The biochemical network can further one or more of an exogenouspolypeptide having thioesterase activity, a polypeptide having aldehydedehydrogenase activity, or a polypeptide having butanal dehydrogenaseactivity, wherein the polypeptide having thioesterase activity, apolypeptide having aldehyde dehydrogenase activity, or a polypeptidehaving butanal dehydrogenase activity enzymatically produce pentanoateas a C5 precursor molecule.

Also, described herein is a biochemical network comprising at least oneexogenous nucleic acid encoding a polypeptide having alcoholO-acetyltransferase activity and a polypeptide having monooxygenaseactivity, wherein the biochemical network produces pentanoic acid pentylester. The biochemical network can further include an esterase, whereinthe esterase enzymatically converts pentanoic acid pentyl ester to5-hydroxypentanoate and/or 1,5-pentanediol.

The biochemical network can further include at least one exogenousnucleic acid encoding a polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity, wherein the polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity enzymatically produce C5 precursor molecules such aspentanoyl-CoA. The biochemical network can further include one or moreof an exogenous a polypeptide having aldehyde dehydrogenase activity, apolypeptide having alcohol dehydrogenase activity, a polypeptide havingbutanal dehydrogenase activity, a polypeptide having carboxylatereductase activity or a polypeptide having thioesterase activity,wherein the polypeptide having aldehyde dehydrogenase activity, apolypeptide having alcohol dehydrogenase activity, a polypeptide havingbutanal dehydrogenase activity, a polypeptide having carboxylatereductase activity or a polypeptide having thioesterase activityenzymatically produce pentanol as a C5 precursor molecule.

A biochemical network producing 5-hydroxypentanoate can further includeone or more of a polypeptide having monooxygenase activity, apolypeptide having alcohol dehydrogenase activity, a polypeptide havingaldehyde dehydrogenase activity, a polypeptide having 7-oxohexanoatedehydrogenase activity, a polypeptide having 6-oxohexanoatedehydrogenase activity, a polypeptide having 5-hydroxypentanoatedehydrogenase activity, a polypeptide having 4-hydroxybutyratedehydrogenase activity or a polypeptide having 6-hydroxyhexanoatedehydrogenase activity, wherein the polypeptide having monooxygenaseactivity, a polypeptide having alcohol dehydrogenase activity, apolypeptide having aldehyde dehydrogenase activity, a polypeptide having7-oxohexanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having5-hydroxypentanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity or a polypeptide having6-hydroxyhexanoate dehydrogenase activity enzymatically convert5-hydroxyheptanoate to glutaric acid or glutarate semialdehyde.

A biochemical network producing 5-hydroxypentanoate can further includeone or more of a polypeptide having ω-transaminase activity, apolypeptide having 6-hydroxyhexanoate dehydrogenase activity, apolypeptide having 5-hydroxybutanoate dehydrogenase activity, apolypeptide having 4-hydroxybutyrate dehydrogenase activity and apolypeptide having alcohol dehydrogenase activity, wherein thepolypeptide having ω-transaminase activity, a polypeptide having6-hydroxyhexanoate dehydrogenase activity, a polypeptide having5-hydroxybutanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity and a polypeptide havingalcohol dehydrogenase activity enzymatically convert 5-hydroxypentanoateto 5-aminopentanoate.

A biochemical network producing 5-aminopentanoate, 5-hydroxypentanoate,glutarate semialdehyde, or 1,5-pentanediol can further include one ormore of a polypeptide having carboxylate reductase activity, apolypeptide having ω-transaminase activity, a polypeptide havingdeacetylase activity, a polypeptide having N-acetyl transferaseactivity, or a polypeptide having alcohol dehydrogenase activity,wherein the a polypeptide having carboxylate reductase activity, apolypeptide having ω-transaminase activity, a polypeptide havingdeacetylase activity, a polypeptide having N-acetyl transferaseactivity, or a polypeptide having alcohol dehydrogenase activity,enzymatically convert 5-aminopentanoate, 5-hydroxypentanoate, glutaratesemialdehyde, or 1,5-pentanediol to cadaverine.

A biochemical network producing 5-hydroxypentanoate can further includeone or more of a polypeptide having carboxylate reductase activity and apolypeptide having alcohol dehydrogenase activity, wherein thepolypeptide having carboxylate reductase activity and a polypeptidehaving alcohol dehydrogenase activity enzymatically convert5-hydroxypentanoate to 1,5-pentanediol.

Also, described herein is a means for obtaining 5-hydroxypentanoateusing (i) a polypeptide having fatty acid O-methyltransferase activityand a polypeptide having monooxygenase activity and (ii) a polypeptidehaving demethylase activity or a polypeptide having esterase activity.The means can further include means for converting 5-hydroxypentanoateto at least one of glutaric acid, 5-aminopentanoate, cadaverine,5-hydroxypentanoate, and 1,5-pentanediol. The means can include apolypeptide having aldehyde dehydrogenase activity, a polypeptide having7-oxohexanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having5-hydroxypentanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity or a polypeptide having6-hydroxyhexanoate dehydrogenase activity.

Also, described herein is a means for obtaining 5-hydroxypentanoateusing (i) a polypeptide having alcohol O-acetyltransferase and apolypeptide having monooxygenase activity and (ii) a polypeptide havingdemethylase activity or a polypeptide having esterase activity. Themeans can further include means for converting 5-hydroxypentanoate to atleast one of glutaric acid, 5-aminopentanoate, cadaverine,5-hydroxypentanoate, and 1,5-pentanediol. The means can include apolypeptide having aldehyde dehydrogenase activity, a polypeptide having7-oxohexanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having5-hydroxypentanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity or a polypeptide having6-hydroxyhexanoate dehydrogenase activity.

Also described herein is (i) a step for obtaining 5-hydroxypentanoateusing a polypeptide having alcohol O-acetyltransferase, a polypeptidehaving monooxygenase activity, and a polypeptide having demethylaseactivity or a polypeptide having esterase activity, and (ii) a step forobtaining glutaric acid, 5-aminopentanoate, glutarate semialdehyde1,5-pentanediol, or cadaverine using a polypeptide having carboxylatereductase activity, a polypeptide having alcohol dehydrogenase activity,a polypeptide having ω-transaminase activity, a polypeptide havingdeacetylase activity, a polypeptide having N-acetyl transferaseactivity, a polypeptide having 6-hydroxyhexanoate dehydrogenaseactivity, a polypeptide having 5-hydroxybutanoate dehydrogenaseactivity, a polypeptide having 4-hydroxybutyrate dehydrogenase activity,a polypeptide having aldehyde dehydrogenase activity, a polypeptidehaving 7-oxohexanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having5-hydroxypentanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity or a polypeptide having6-hydroxyhexanoate dehydrogenase activity,

In another aspect, this document features a composition comprising5-hydroxypentanoate and a polypeptide having alcoholO-acetyltransferase, a polypeptide having monooxygenase activity, and apolypeptide having demethylase activity or a polypeptide having esteraseactivity complex. The composition can be cellular. The composition canfurther include a polypeptide having carboxylate reductase activity, apolypeptide having alcohol dehydrogenase activity, a polypeptide havingω-transaminase activity, a polypeptide having deacetylase activity, apolypeptide having N-acetyl transferase activity, a polypeptide having6-hydroxyhexanoate dehydrogenase activity, a polypeptide having5-hydroxybutanoate dehydrogenase activity, a polypeptide having4-hydroxybutyrate dehydrogenase activity, a polypeptide having aldehydedehydrogenase activity, a polypeptide having 7-oxohexanoatedehydrogenase activity, a polypeptide having 6-oxohexanoatedehydrogenase activity, a polypeptide having 5-hydroxypentanoatedehydrogenase activity, a polypeptide having 4-hydroxybutyratedehydrogenase activity or a polypeptide having 6-hydroxyhexanoatedehydrogenase activity, and at least one of glutaric acid,5-aminopentanoic acid, cadaverine, 5-hydroxypentanoate, and1,5-pentanediol. The composition can be cellular.

The reactions of the pathways described herein can be performed in oneor more cell (e.g., host cell) strains (a) naturally expressing one ormore relevant enzymes, (b) genetically engineered to express one or morerelevant enzymes, or (c) naturally expressing one or more relevantenzymes and genetically engineered to express one or more relevantenzymes. Alternatively, relevant enzymes can be extracted from of theabove types of host cells and used in a purified or semi-purified form.Extracted enzymes can optionally be immobilized to the floors and/orwalls of appropriate reaction vessels. Moreover, such extracts includelysates (e.g. cell lysates) that can be used as sources of relevantenzymes. In the methods provided by the document, all the steps can beperformed in cells (e.g., host cells), all the steps can be performedusing extracted enzymes, or some of the steps can be performed in cellsand others can be performed using extracted enzymes.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein including GenBank and NCBI submissions with accessionnumbers are incorporated by reference in their entirety. In case ofconflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and the drawings, and from the claims. The word “comprising”in the claims may be replaced by “consisting essentially of” or with“consisting of,” according to standard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of exemplary biochemical pathways leading topentanoyl-CoA using NADH-dependent enzymes and acetyl-CoA andpropanoyl-CoA as central metabolites.

FIG. 2 is a schematic of exemplary biochemical pathways leading topentanoyl-CoA using NADPH-dependent enzymes and with acetyl-CoA andpropanoyl-CoA as central metabolites.

FIG. 3 is a schematic of exemplary biochemical pathways leading topentanoate and pentanol using pentanoyl-CoA as a central precursor.

FIG. 4 is a schematic of an exemplary biochemical pathway leading toglutaric acid using 5-hydroxypentanoate as a central precursor.

FIG. 5 is a schematic of an exemplary biochemical pathway leading to5-aminopentanoate using 5-hydroxypentanoate as a central precursor.

FIG. 6 is a schematic of exemplary biochemical pathways leading tocadaverine using 5-aminopentanoate, 5-hydroxypentanoate, glutaratesemialdehyde (also known as 5-oxopentanoic acid), or 1,5-pentanediol asa central precursor.

FIG. 7 is a schematic of exemplary biochemical pathways leading to5-hydroxypentanoate via ester intermediates using pentanoate orpentanoyl-CoA. FIG. 7 also contains an exemplary biochemical pathwayleading to 5-hydroxypentanoate using 1,5-pentanediol as a centralprecursor.

FIG. 8 is a schematic of exemplary biochemical pathways leading to 1,5pentanediol using 5-hydroxypentanoate or pentanoyl-CoA as a centralprecursor.

FIG. 9 is a schematic of exemplary biochemical pathways leading topropanoyl-CoA from central metabolites.

FIG. 10 contains the amino acid sequences of an Escherichia colithioesterase encoded by tesB (see GenBank Accession No. AAA24665.1, SEQID NO: 1), a Mycobacterium marinum carboxylate reductase (see GenbankAccession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatiscarboxylate reductase (see Genbank Accession No. ABK71854.1, SEQ ID NO:3), a Segniliparus rugosus carboxylate reductase (see Genbank AccessionNo. EFV11917.1, SEQ ID NO: 4), a Mycobacterium smegmatis carboxylatereductase (see Genbank Accession No. ABK75684.1, SEQ ID NO: 5), aMycobacterium massiliense carboxylate reductase (see Genbank AccessionNo. EIV11143.1, SEQ ID NO: 6), a Segniliparus rotundus carboxylatereductase (see Genbank Accession No. ADG98140.1, SEQ ID NO: 7), aChromobacterium violaceum ω-transaminase (see Genbank Accession No.AAQ59697.1, SEQ ID NO: 8), a Pseudomonas aeruginosa ω-transaminase (seeGenbank Accession No. AAG08191.1, SEQ ID NO: 9), a Pseudomonas syringaeω-transaminase (see Genbank Accession No. AAY39893.1, SEQ ID NO: 10), aRhodobacter sphaeroides ω-transaminase (see Genbank Accession No.ABA81135.1, SEQ ID NO: 11), an Escherichia coli ω-transaminase (seeGenbank Accession No. AAA57874.1, SEQ ID NO: 12), a Vibrio fluvialisω-transaminase (See Genbank Accession No. AEA39183.1, SEQ ID NO: 13); aPolaromonas sp. JS666 monooxygenase (see Genbank Accession No.ABE47160.1, SEQ ID NO:14), a Mycobacterium sp. HXN-1500 monooxygenase(see Genbank Accession No. CAH04396.1, SEQ ID NO:15), a Mycobacteriumaustroafricanum monooxygenase (see Genbank Accession No. ACJ06772.1, SEQID NO:16), a Polaromonas sp. JS666 oxidoreductase (see Genbank AccessionNo. ABE47159.1, SEQ ID NO:17), a Mycobacterium sp. HXN-1500oxidoreductase (see Genbank Accession No. CAH04397.1, SEQ ID NO:18), aPolaromonas sp. JS666 ferredoxin (see Genbank Accession No. ABE47158.1,SEQ ID NO:19), a Mycobacterium sp. HXN-1500 ferredoxin (see GenbankAccession No. CAH04398.1, SEQ ID NO:20), Bacillus subtilisphosphopantetheinyl transferase (see Genbank Accession No. CAA44858.1,SEQ ID NO:21), a Nocardia sp. NRRL 5646 phosphopantetheinyl transferase(see Genbank Accession No. ABI83656.1, SEQ ID NO:22), a Mycobacteriummarinum fatty acid O-methyltransferase (GenBank Accession No.ACC41782.1; SEQ ID NO:23), a Mycobacterium smegmatis str. MC2 fatty acidO-methyltransferase (GenBank Accession No. ABK73223.1; SEQ ID NO:24), aPseudomonas putida fatty acid O-methyltransferase (GenBank Accession No.CAA39234.1; SEQ ID NO:25), a Saccharomyces cerevisiae alcoholO-acetyltransferase (Genbank Accession No: CAA85138.1, SEQ ID NO: 26), aPseudomonas fluorescens carboxylesterase (Genbank Accession No.AAC60471.2, SEQ ID NO: 27), a Pseudomonas putida alkane 1-monooxygenase(Genbank Accession No. CAB51047.1, SEQ ID NO: 28), a Candida maltosecytochrome P450 (Genbank Accession No: BAA00371.1, SEQ ID NOs: 29), aSalmonella enterica subsp. enterica serovar Typhimurium butanaldehydrogenase (GenBank Accession No. AAD39015, SEQ ID NO:30), aSphingomonas paucimobilis demethylase (GenBank Accession No. BAD61059.1and GenBank Accession No. BAC59257.1, SEQ ID NOs: 31 and 32,respectively), a Lactobacillus brevis thioesterase (GenBank AccessionNo. ABJ63754.1, SEQ ID NO:33), and a Lactobacillus plantarumthioesterase (GenBank Accession No. CCC58182.1, SEQ ID NO:34).

FIG. 11 is a bar graph of the change in absorbance at 340 nm after 20min, which is a measure of the consumption of NADPH and activity ofcarboxylate reductases for converting pentanoic acid to pentanalrelative to the empty vector control.

FIG. 12 is a bar graph summarizing the change in absorbance at 340 nmafter 20 minutes, which is a measure of the consumption of NADPH andactivity of five carboxylate reductase preparations in enzyme onlycontrols (no substrate).

FIG. 13 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity of six ω-transaminase preparations for converting5-aminopentanol to 5-oxopentanol relative to the empty vector control.

FIG. 14 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof five carboxylate reductase preparations for converting5-hydroxypentanoate to 5-hydroxypentanal relative to the empty vectorcontrol.

FIG. 15 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity of five ω-transaminase preparations for convertingN5-acetyl-1,5-diaminopentane to N5-acetyl-5-aminopentanal relative tothe empty vector control.

FIG. 16 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and activity ofa carboxylate reductase preparation for converting glutaratesemialdehyde to pentanedial relative to the empty vector control.

FIG. 17 is a bar graph summarizing the percent conversion of pyruvate toL-alanine (mol/mol) as a measure of the ω-transaminase activity of theenzyme only controls (no substrate).

FIG. 18 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity of one ω-transaminase preparation for converting5-aminopentanoate to glutarate semialdehyde relative to the empty vectorcontrol.

FIG. 19 is a bar graph of the percent conversion after 4 hours ofL-alanine to pyruvate (mol/mol) as a measure of the ω-transaminaseactivity of one ω-transaminase preparation for converting glutaratesemialdehyde to 5-aminopentanoate relative to the empty vector control.

FIG. 20 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity of four ω-transaminase preparations for converting cadaverineto 5-aminopentanal relative to the empty vector control.

DETAILED DESCRIPTION

This document provides enzymes, non-natural pathways, cultivationstrategies, feedstocks, central precursors, host microorganisms andattenuations to the host's biochemical network, which generate a sevencarbon chain aliphatic backbone (which can be bound to a coenzyme Amoiety) from central metabolites in which one or two terminal functionalgroups may be formed leading to the synthesis of one or more of glutaricacid, 5-hydroxypentanoate, 5-aminopentanoate, cadaverine or1,5-pentanediol (referred to as “C5 building blocks” herein). As usedherein, the term “central precursor” is used to denote any metabolite inany metabolic pathway shown herein leading to the synthesis of a C5building block. The term “central metabolite” is used herein to denote ametabolite that is produced in all microorganisms to support growth.

Host microorganisms described herein can include endogenous pathwaysthat can be manipulated such that one or more C5 building blocks orcentral precursors thereof can be produced. In an endogenous pathway,the host microorganism naturally expresses all of the enzymes catalyzingthe reactions within the pathway. A host microorganism containing anengineered pathway does not naturally express all of the enzymescatalyzing the reactions within the pathway but has been engineered suchthat all of the enzymes within the pathway are expressed in the host.

The term “exogenous” as used herein with reference to a nucleic acid (ora protein) and a host refers to a nucleic acid that does not occur in(and cannot be obtained from) a cell of that particular type as it isfound in nature or a protein encoded by such a nucleic acid. Thus, anon-naturally-occurring nucleic acid is considered to be exogenous to ahost once in the host. It is important to note thatnon-naturally-occurring nucleic acids can contain nucleic acidsubsequences or fragments of nucleic acid sequences that are found innature provided the nucleic acid as a whole does not exist in nature.For example, a nucleic acid molecule containing a genomic DNA sequencewithin an expression vector is non-naturally-occurring nucleic acid, andthus is exogenous to a host cell once introduced into the host, sincethat nucleic acid molecule as a whole (genomic DNA plus vector DNA) doesnot exist in nature. Thus, any vector, autonomously replicating plasmid,or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a wholedoes not exist in nature is considered to be non-naturally-occurringnucleic acid. It follows that genomic DNA fragments produced by PCR orrestriction endonuclease treatment as well as cDNAs are considered to benon-naturally-occurring nucleic acid since they exist as separatemolecules not found in nature. It also follows that any nucleic acidcontaining a promoter sequence and polypeptide-encoding sequence (e.g.,cDNA or genomic DNA) in an arrangement not found in nature isnon-naturally-occurring nucleic acid. A nucleic acid that isnaturally-occurring can be exogenous to a particular host microorganism.For example, an entire chromosome isolated from a cell of yeast x is anexogenous nucleic acid with respect to a cell of yeast y once thatchromosome is introduced into a cell of yeast y.

In contrast, the term “endogenous” as used herein with reference to anucleic acid (e.g., a gene) (or a protein) and a host refers to anucleic acid (or protein) that does occur in (and can be obtained from)that particular host as it is found in nature. Moreover, a cell“endogenously expressing” a nucleic acid (or protein) expresses thatnucleic acid (or protein) as does a host of the same particular type asit is found in nature. Moreover, a host “endogenously producing” or that“endogenously produces” a nucleic acid, protein, or other compoundproduces that nucleic acid, protein, or compound as does a host of thesame particular type as it is found in nature.

For example, depending on the host and the compounds produced by thehost, one or more of the following polypeptides may be expressed in thehost in addition to a polypeptide having fatty acid O-methyltransferaseactivity or a polypeptide having alcohol O-acetyltransferase activity: amonooxygenase, an esterase, a demethylase, a β-ketothiolase, anacetyl-CoA carboxylase, a β-ketoacyl-[acp] synthase, a 3-hydroxyacyl-CoAdehydrogenase, a 3-oxoacyl-CoA reductase, an enoyl-CoA hydratase, atrans-2-enoyl-CoA reductase, a thioesterase, an aldehyde dehydrogenase,a butanal dehydrogenase, a monooxygenase in, for example, the CYP4F3Bfamily, an alcohol dehydrogenase, a 7-oxoheptanoate dehydrogenase, a6-oxohexanoate dehydrogenase, a 5-oxopentanoate dehydrogenase, aω-transaminase, a 6-hydroxyhexanoate dehydrogenase, a5-hydroxypentanoate dehydrogenase, 4-hydroxybutyrate dehydrogenase, acarboxylate reductase, a deacetylase, or an N-acetyl transferase. Inrecombinant hosts expressing a carboxylate reductase, aphosphopantetheinyl transferase also can be expressed as it enhancesactivity of the carboxylate reductase. In recombinant hosts expressing amonooxygenase, an electron transfer chain protein such as anoxidoreductase and/or ferredoxin polypeptide also can be expressed.

In some embodiments, a recombinant host can include at least oneexogenous nucleic acid encoding a polypeptide having (i) fatty acidO-methyltransferase activity or alcohol O-acetyltransferase activity,(ii) monooxygenase activity, and (iii) esterase or demethylase activity.

In some embodiments, a recombinant host can include at least oneexogenous nucleic acid encoding a polypeptide having fatty acidO-methyltransferase and a polypeptide having monooxygenase activity,wherein the host produces 5-hydroxypentanoate methyl ester. Such a hostfurther can include a polypeptide having demethylase or esteraseactivity and further produce 5-hydroxypentanoate. In some embodiments,the recombinant host also can include at least one exogenous nucleicacid encoding a polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity to produce C5 precursor molecules such as pentanoyl-CoA. Such ahost further can include one or more of (e.g., two or three of) anexogenous polypeptide having thioesterase activity, a polypeptide havingaldehyde dehydrogenase activity, or a polypeptide having butanaldehydrogenase activity, and produce pentanoate as a C5 precursormolecule.

In some embodiments, a recombinant host can include at least oneexogenous nucleic acid encoding a polypeptide having alcoholO-acetyltransferase activity and a polypeptide having monooxygenaseactivity, wherein the host produces pentanoic acid pentyl ester. Such ahost further can include a polypeptide having esterase activity andfurther produce 5-hydroxypentanoate and/or 1,5-pentanediol. In someembodiments, the recombinant host also can include at least oneexogenous nucleic acid encoding a polypeptide having β-ketothiolaseactivity or a polypeptide having acetyl-CoA carboxylase activity and apolypeptide having β-ketoacyl-[acp] synthase activity, a polypeptidehaving 3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity to produce C5 precursor molecules such as pentanoyl-CoA. Such ahost further can include one or more of (e.g., two or three of) anexogenous polypeptide having aldehyde dehydrogenase activity, apolypeptide having alcohol dehydrogenase activity, a polypeptide havingbutanal dehydrogenase activity, a polypeptide having carboxylatereductase activity or a polypeptide having thioesterase activity andproduce pentanol as a C5 precursor molecule.

A recombinant host producing 5-hydroxypentanoate further can include oneor more polypeptides having an activity of a monooxygenase (e.g., in theCYP4F3B family) an alcohol dehydrogenase, an aldehyde dehydrogenase, a6-hydroxyhexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, a6-oxohexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a4-hydroxybutyrate dehydrogenase or a 5-oxopentanoate dehydrogenase, andproduce glutaric acid. For example, a recombinant host further caninclude a polypeptide having monooxygenase activity and produce glutaricacid. As another example, a recombinant host further can include apolypeptide having the activity of (i) an alcohol dehydrogenase, a6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase,or a 4-hydroxybutyrate dehydrogenase or (ii) an aldehyde dehydrogenase,a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, or a5-oxopentanoate dehydrogenase, and produce glutaric acid.

A recombinant host producing 5-hydroxypentanoate further can include oneor more polypeptides having the activity of a transaminase, a6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a4-hydroxybutyrate dehydrogenase and an alcohol dehydrogenase, andproduce 5-aminopentanoate. For example, a recombinant host producing5-hydroxypentanoate further can include a polypeptide havingω-transaminase activity and either a polypeptide having6-hydroxyhexanoate dehydrogenase activity or having alcoholdehydrogenase activity.

A recombinant host producing 5-aminopentanoate, 5-hydroxypentanoate,glutarate semialdehyde or 1,5-pentanediol further can include one ormore of a polypeptide having carboxylate reductase activity, apolypeptide having ω-transaminase activity, a polypeptide havingdeacetylase activity, a polypeptide having N-acetyl transferaseactivity, or a polypeptide having alcohol dehydrogenase activity, andproduce cadaverine. In some embodiments, a recombinant host further caninclude each of a polypeptide having carboxylate reductase activity, apolypeptide having ω-transaminase activity, a polypeptide havingdeacetylase activity, and a polypeptide having N-acetyl transferaseactivity. In some embodiments, a recombinant host further can include apolypeptide having carboxylate reductase activity and a polypeptidehaving ω-transaminase activity. In some embodiments, a recombinant hostfurther can include a polypeptide having carboxylate reductase activity,a polypeptide having ω-transaminase activity, and a polypeptide havingalcohol dehydrogenase activity. In the embodiments in which therecombinant host produces 5-aminopentanoate, an additionalω-transaminase may not be necessary to produce cadaverine. In someembodiments, the host includes a second exogenous polypeptide havingω-transaminase activity that differs from the first exogenouspolypeptide having ω-transaminase activity.

A recombinant host producing 5-hydroxypentanoic acid further can includeone or more of a polypeptide having carboxylate reductase activity and apolypeptide having alcohol dehydrogenase activity, and produce1,5-pentanediol.

Within an engineered pathway, the enzymes can be from a single source,i.e., from one species or genus, or can be from multiple sources, i.e.,different species or genera. Nucleic acids encoding the enzymesdescribed herein have been identified from various organisms and arereadily available in publicly available databases such as GenBank orEMBL. In recombinant hosts containing an exogenous enzyme, the hostscontain an exogenous nucleic acid encoding the enzyme.

Any of the enzymes described herein that can be used for production ofone or more C5 building blocks can have at least 70% sequence identity(homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of thecorresponding wild-type enzyme. It will be appreciated that the sequenceidentity can be determined on the basis of the mature enzyme (e.g., withany signal sequence removed) or on the basis of the immature enzyme(e.g., with any signal sequence included). It also will be appreciatedthat the initial methionine residue may or may not be present on any ofthe enzyme sequences described herein.

For example, a polypeptide having thioesterase activity described hereincan have at least 70% sequence identity (homology) (e.g., at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) tothe amino acid sequence of an Escherichia coli thioesterase encoded bytesB (see GenBank Accession No. AAA24665.1, SEQ ID NO: 1), to the aminoacid sequence of a Lactobacillus brevis thioesterase (GenBank AccessionNo. ABJ63754.1, SEQ ID NO:33) or a Lactobacillus plantarum thioesterase(GenBank Accession No. CCC58182.1, SEQ ID NO:34). See FIG. 10.

For example, a polypeptide having carboxylate reductase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Mycobacterium marinum(see Genbank Accession No. ACC40567.1, SEQ ID NO: 2), a Mycobacteriumsmegmatis (see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), aSegniliparus rugosus (see Genbank Accession No. EFV11917.1, SEQ ID NO:4), a Mycobacterium smegmatis (see Genbank Accession No. ABK75684.1, SEQID NO: 5), a Mycobacterium massiliense (see Genbank Accession No.EIV11143.1, SEQ ID NO: 6), or a Segniliparus rotundus (see GenbankAccession No. ADG98140.1, SEQ ID NO: 7) carboxylate reductase. See, FIG.10.

For example, a polypeptide having ω-transaminase activity describedherein can have at least 70% sequence identity (homology) (e.g., atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) to the amino acid sequence of a Chromobacterium violaceum (seeGenbank Accession No. AAQ59697.1, SEQ ID NO: 8), a Pseudomonasaeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO: 9), aPseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ ID NO:10), a Rhodobacter sphaeroides (see Genbank Accession No. ABA81135.1,SEQ ID NO: 11), an Escherichia coli (see Genbank Accession No.AAA57874.1, SEQ ID NO: 12), or a Vibrio fluvialis (see Genbank AccessionNo. AEA39183.1, SEQ ID NO: 13) ω-transaminase. Some of theseω-transaminases are diamine ω-transaminases. See, FIG. 10.

For example, a polypeptide having monooxygenase activity describedherein can have at least 70% sequence identity (homology) (e.g., atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) to the amino acid sequence of a Polaromonas sp. JS666monooxygenase (see Genbank Accession No. ABE47160.1, SEQ ID NO: 14), aMycobacterium sp. HXN-1500 monooxygenase (see Genbank Accession No.CAH04396.1, SEQ ID NO:15), or a Mycobacterium austroafricanummonooxygenase (See Genbank Accession No. ACJ06772.1, SEQ ID NO:16). See,FIG. 10.

For example, a polypeptide having oxidoreductase activity describedherein can have at least 70% sequence identity (homology) (e.g., atleast 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,or 100%) to the amino acid sequence of a Polaromonas sp. JS666oxidoreductase (see Genbank Accession No. ABE47159.1, SEQ ID NO: 17) ora Mycobacterium sp. HXN-1500 oxidoreductase (see Genbank Accession No.CAH04397.1, SEQ ID NO: 18). See, FIG. 10.

For example, a polypeptide having ferredoxin activity described hereincan have at least 70% sequence identity (homology) (e.g., at least 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) tothe amino acid sequence of a Polaromonas sp. JS666 ferredoxin (seeGenbank Accession No. ABE47158.1, SEQ ID NO: 19) or a Mycobacterium sp.HXN-1500 ferredoxin (see Genbank Accession No. CAH04398.1, SEQ ID NO:20). See, FIG. 10.

For example, a polypeptide having phosphopantetheinyl transferaseactivity described herein can have at least 70% sequence identity(homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Bacillussubtilis phosphopantetheinyl transferase (see Genbank Accession No.CAA44858.1, SEQ ID NO: 21) or a Nocardia sp. NRRL 5646phosphopantetheinyl transferase (see Genbank Accession No. ABI83656.1,SEQ ID NO: 22). See FIG. 10.

For example, a polypeptide having fatty acid O-methyltransferaseactivity described herein can have at least 70% sequence identity(homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of aMycobacterium marinum (see GenBank Accession No. ACC41782.1, SEQ ID NO:23), a Mycobacterium smegmatis (see GenBank Accession No. ABK73223.1,SEQ ID NO: 24), or a Pseudomonas putida (see GenBank Accession No.CAA39234.1, SEQ ID NO: 25) methyltransferase. See FIG. 10.

For example, a polypeptide having alcohol O-acetyltransferase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Saccharomycescerevisiae (see GenBank Accession No. CAA85138.1, SEQ ID NO: 26) alcoholO-acetyltransferase. See FIG. 10.

For example, a polypeptide having esterase activity described herein canhave at least 70% sequence identity (homology) (e.g., at least 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to theamino acid sequence of a Pseudomonas fluorescens (see GenBank AccessionNo. AAC60471.2, SEQ ID NO: 27) esterase. See FIG. 10.

For example, a polypeptide having alkane 1-monooxygenase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Pseudomonas putidaalkane 1-monooxygenase (see Genbank Accession No. CAB51047.1, SEQ ID NO:28).

For example, a polypeptide having cytochrome P450 monooxygenase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Candida maltosecytochrome P450 (see Genbank Accession No: BAA00371.1, SEQ ID NOs: 29).

For example, a polypeptide having butanal dehydrogenase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Salmonella entericasubsp. enterica serovar Typhimurium butanal dehydrogenase (see GenBankAccession No. AAD39015, SEQ ID NO:30).

For example, a polypeptide having syringate O-demethylase activitydescribed herein can have at least 70% sequence identity (homology)(e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100%) to the amino acid sequence of a Sphingomonaspaucimobilis demethylase (see, GenBank Accession No. BAD61059.1 andGenBank Accession No. BAC59257.1, SEQ ID NOs: 31 and 32, respectively).

The percent identity (homology) between two amino acid sequences can bedetermined as follows. First, the amino acid sequences are aligned usingthe BLAST 2 Sequences (Bl2seq) program from the stand-alone version ofBLASTZ containing BLASTP version 2.0.14. This stand-alone version ofBLASTZ can be obtained from Fish & Richardson's web site (e.g.,www.fr.com/blast/) or the U.S. government's National Center forBiotechnology Information web site (www.ncbi.nlm.nih.gov). Instructionsexplaining how to use the Bl2seq program can be found in the readme fileaccompanying BLASTZ. Bl2seq performs a comparison between two amino acidsequences using the BLASTP algorithm. To compare two amino acidsequences, the options of Bl2seq are set as follows: -i is set to a filecontaining the first amino acid sequence to be compared (e.g.,C:\seq1.txt); -j is set to a file containing the second amino acidsequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o isset to any desired file name (e.g., C:\output.txt); and all otheroptions are left at their default setting. For example, the followingcommand can be used to generate an output file containing a comparisonbetween two amino acid sequences: C:\Bl2seq -i c:\seq1.txt -jc:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequencesshare homology (identity), then the designated output file will presentthose regions of homology as aligned sequences. If the two comparedsequences do not share homology (identity), then the designated outputfile will not present aligned sequences. Similar procedures can befollowing for nucleic acid sequences except that blastn is used.

Once aligned, the number of matches is determined by counting the numberof positions where an identical amino acid residue is presented in bothsequences. The percent identity (homology) is determined by dividing thenumber of matches by the length of the full-length polypeptide aminoacid sequence followed by multiplying the resulting value by 100. It isnoted that the percent identity (homology) value is rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded upto 78.2. It also is noted that the length value will always be aninteger.

It will be appreciated that a number of nucleic acids can encode apolypeptide having a particular amino acid sequence. The degeneracy ofthe genetic code is well known to the art; i.e., for many amino acids,there is more than one nucleotide triplet that serves as the codon forthe amino acid. For example, codons in the coding sequence for a givenenzyme can be modified such that optimal expression in a particularspecies (e.g., bacteria or fungus) is obtained, using appropriate codonbias tables for that species.

Functional fragments of any of the enzymes described herein can also beused in the methods of the document. The term “functional fragment” asused herein refers to a peptide fragment of a protein that is shorterthan the full-length immature protein and has at least 25% (e.g., atleast: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 91%; 92%; 93%; 94%;95%; 96%; 97%; 98%; 99%; 100%; or even greater than 100%) of theactivity of the corresponding mature, full-length, wild-type protein.The functional fragment can generally, but not always, be comprised of acontinuous region of the protein, wherein the region has functionalactivity.

This document also provides (i) functional variants of the enzymes usedin the methods of the document and (ii) functional variants of thefunctional fragments described above. Functional variants of the enzymesand functional fragments can contain additions, deletions, orsubstitutions relative to the corresponding wild-type sequences. Enzymeswith substitutions will generally have not more than 50 (e.g., not morethan one, two, three, four, five, six, seven, eight, nine, ten, 12, 15,20, 25, 30, 35, 40, or 50) amino acid substitutions (e.g., conservativesubstitutions). This applies to any of the enzymes described herein andfunctional fragments. A conservative substitution is a substitution ofone amino acid for another with similar characteristics. Conservativesubstitutions include substitutions within the following groups: valine,alanine and glycine; leucine, valine, and isoleucine; aspartic acid andglutamic acid; asparagine and glutamine; serine, cysteine, andthreonine; lysine and arginine; and phenylalanine and tyrosine. Thenonpolar hydrophobic amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan and methionine. The polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine and glutamine. The positively charged (basic) aminoacids include arginine, lysine and histidine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid. Anysubstitution of one member of the above-mentioned polar, basic or acidicgroups by another member of the same group can be deemed a conservativesubstitution. By contrast, a nonconservative substitution is asubstitution of one amino acid for another with dissimilarcharacteristics.

Deletion variants can lack one, two, three, four, five, six, seven,eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsegments (of two or more amino acids) or non-contiguous single aminoacids. Additions (addition variants) include fusion proteins containing:(a) any of the enzymes described herein or a fragment thereof; and (b)internal or terminal (C or N) irrelevant or heterologous amino acidsequences. In the context of such fusion proteins, the term“heterologous amino acid sequences” refers to an amino acid sequenceother than (a). A heterologous sequence can be, for example a sequenceused for purification of the recombinant protein (e.g., FLAG,polyhistidine (e.g., hexahistidine), hemagglutinin (HA),glutathione-S-transferase (GST), or maltosebinding protein (MBP)).Heterologous sequences also can be proteins useful as detectablemarkers, for example, luciferase, green fluorescent protein (GFP), orchloramphenicol acetyl transferase (CAT). In some embodiments, thefusion protein contains a signal sequence from another protein. Incertain host cells (e.g., yeast host cells), expression and/or secretionof the target protein can be increased through use of a heterologoussignal sequence. In some embodiments, the fusion protein can contain acarrier (e.g., KLH) useful, e.g., in eliciting an immune response forantibody generation) or ER or Golgi apparatus retention signals.Heterologous sequences can be of varying length and in some cases can bea longer sequences than the full-length target proteins to which theheterologous sequences are attached.

Engineered hosts can naturally express none or some (e.g., one or more,two or more, three or more, four or more, five or more, or six or more)of the enzymes of the pathways described herein. Thus, a pathway withinan engineered host can include all exogenous enzymes, or can includeboth endogenous and exogenous enzymes. Endogenous genes of theengineered hosts also can be disrupted to prevent the formation ofundesirable metabolites or prevent the loss of intermediates in thepathway through other enzymes acting on such intermediates. Engineeredhosts can be referred to as recombinant hosts or recombinant host cells.As described herein recombinant hosts can include nucleic acids encodingone or more of a polypeptide having fatty acid O-methyltransferaseactivity, a polypeptide having alcohol O-acetyltransferase activity, apolypeptide having dehydrogenase activity, a polypeptide havingβ-ketothiolase activity, a polypeptide having β-ketoacyl-[acp] synthaseactivity, a polypeptide having carboxylase activity, a polypeptidehaving reductase activity, a polypeptide having hydratase activity, apolypeptide having thioesterase activity, a polypeptide havingmonooxygenase activity, a polypeptide having demethylase activity, apolypeptide having esterase activity, or a polypeptide havingtransaminase activity as described herein.

In addition, the production of one or more C5 building blocks can beperformed in vitro using the isolated enzymes described herein, using alysate (e.g., a cell lysate) from a host microorganism as a source ofthe enzymes, or using a plurality of lysates from different hostmicroorganisms as the source of the enzymes.

Biosynthetic Methods

The present document provides methods of producing a (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester. As used herein, the term (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester refers to a compound having the followingformula:

As used herein, the term “C₃₋₈ hydroxyalkyl” refers to a saturatedhydrocarbon group that may be straight-chain or branched, and issubstituted by at least one hydroxyl (i.e., hydroxy or OH) group. Insome embodiments, the C₃₋₈ hydroxyalkyl refers to refers to a saturatedhydrocarbon group that may be straight-chain or branched, and issubstituted by at least one terminal hydroxyl (OH) group. In someembodiments, the alkyl group contains 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5to 8, 5 to 7, 5 to 6, 6 to 8, 6 to 7, or 7 to 8 carbon atoms. In someembodiments, the C₃₋₈ hydroxyalkyl is a group of the following formula:

In some embodiments, the method comprises:

a) enzymatically converting a C₄₋₉ carboxylic acid to a (C₃₋₈alkyl)-C(═O)OCH₃ ester; and

b) enzymatically converting the (C₃₋₈ alkyl)-C(═O)OCH₃ ester to (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester.

As used herein, the term “C₄₋₉ carboxylic acid” refers to a compoundhaving the formula R—C(═O)OH, wherein R is a refers to a saturatedhydrocarbon group (i.e., an alkyl group) that may be straight-chain orbranched, wherein the compound has from 4 to 9 carbon atoms. In someembodiments, the C₄₋₉ carboxylic acid group contains 4 to 9, 4 to 8, 4to 7, 4 to 6, 4 to 5, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 9, 6 to 8, 6to 7, 7 to 9, 7 to 8, or 8 to 9 carbon atoms. Exemplary C₄₋₉ carboxylicacids include butanoic acid (i.e., butanoate), pentanoic acid (i.e.,pentanoate), hexanoic acid (i.e., hexanoate), heptanoic acid (i.e.,heptanoate), octanoic acid (e.g., octanoate), nonanoic acid (i.e.,nonanoate), 2-methylpentanoic acid (i.e., 2-methylpentanoate),3-methylpentanoic acid (i.e., 3-methylpentanoate), and 4-methylpentanoicacid (i.e., 4-methylpentanoate). In some embodiments, the C₄₋₉carboxylic acid is pentanoic acid (i.e., pentanoate).

As used herein, the term “(C₃₋₈ alkyl)-C(═O)OCH₃ ester” refers to acompound having the following formula:

As used herein, the term “C₃₋₈ alkyl” refers to a saturated hydrocarbongroup that may be straight-chain or branched, having 3 to 8 carbonatoms. In some embodiments, the alkyl group contains 4 to 8, 4 to 7, 4to 6, 4 to 5, 5 to 8, 5 to 7, 5 to 6, 6 to 8, 6 to 7, or 7 to 8, carbonatoms. Example alkyl moieties include n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, iso-pentyl, neo-pentyl, n-hexyl, n-heptyl, andn-octyl. In some embodiments, the C₃₋₈ alkyl is a group of the followingformula:

In some embodiments, the method comprises:

a) enzymatically converting a C₄₋₉ carboxylic acid to a (C₃₋₈alkyl)-C(═O)OCH₃ ester; and

b) enzymatically converting the (C₃₋₈ alkyl)-C(═O)OCH₃ ester to (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester.

In some embodiments, the C₄₋₉ carboxylic acid is enzymatically convertedto the (C₃₋₈ alkyl)-C(═O)OCH₃ ester using a polypeptide having fattyacid O-methyltransferase activity. In some embodiments, the polypeptidehaving fatty acid O-methyltransferase activity has at least 70% sequenceidentity to an amino acid sequence set forth in SEQ ID NO:23, SEQ IDNO:24, or SEQ ID NO:25.

In some embodiments, the (C₃₋₈ alkyl)-C(═O)OCH₃ ester is enzymaticallyconverted to the (C₃₋₈ hydroxyalkyl)-C(═O)OCH₃ ester using a polypeptidehaving monooxygenase activity. In some embodiments, the monooxygenase isclassified under EC 1.14.14.- or EC 1.14.15.-. In some embodiments, themonooxygenase has at least 70% sequence identity to an amino acidsequence set forth in SEQ ID NO:28 or SEQ ID NO:29.

In some embodiments, the C₄₋₉ carboxylic acid is enzymatically producedfrom C₄₋₉ alkanoyl-CoA. As used herein, the term “C₄₋₉ alkanoyl-CoA”refers to a compound having the following formula:

wherein the C₃₋₈ alkyl group is as defined herein. In some embodiments,the C₃₋₈ alkyl is a group of the following formula:

In some embodiments, a polypeptide having thioesterase activityenzymatically produces the C₄₋₉ carboxylic acid from C₄₋₉ alkanoyl-CoA.In some embodiments, the thioesterase has at least 70% sequence identityto the amino acid sequence set forth in SEQ ID NO: 1. In someembodiments, a polypeptide having butanal dehydrogenase activity and apolypeptide having aldehyde dehydrogenase activity enzymaticallyproduces the C₄₋₉ carboxylic acid from the C₄₋₉ alkanoyl-CoA.

In some embodiments, the method of producing a (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester is a method of producing5-hydroxypentanoate methyl ester, said method comprising:

a) enzymatically converting pentanoate to pentanoate methyl ester; and

b) enzymatically converting the pentanoate methyl ester to5-hydroxypentanoate methyl ester.

In some embodiments, pentanoate is enzymatically converted to pentanoatemethyl ester using a polypeptide having fatty acid O-methyltransferaseactivity. In some embodiments, the polypeptide having fatty acidO-methyltransferase activity has at least 70% sequence identity to anamino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:24, or SEQ IDNO:25. In some embodiments, the polypeptide having fatty acidO-methyltransferase activity is classified under EC 12.1.1.15.

In some embodiments, pentanoate methyl ester is enzymatically convertedto 5-hydroxypentanoate methyl ester using a polypeptide havingmonooxygenase activity. In some embodiments, the polypeptide havingmonooxygenase activity has at least 70% sequence identity to an aminoacid sequence set forth in SEQ ID NO:28 or SEQ ID NO:29. In someembodiments, the polypeptide having monooxygenase activity is classifiedunder EC 1.14.14.- or EC 1.14.15.-.

In some embodiments, pentanoate is enzymatically produced frompentanoyl-CoA. In some embodiments, a polypeptide having thioesteraseactivity enzymatically produces pentanoate from pentanoyl-CoA. In someembodiments, the polypeptide having thioesterase activity has at least70% sequence identity to the amino acid sequence set forth in SEQ IDNO:1. In some embodiments, a polypeptide having butanal dehydrogenaseactivity and a polypeptide having aldehyde dehydrogenase activityenzymatically produce pentanoate from pentanoyl-CoA.

The present document further provides methods of producing one or moreterminal hydroxy-substituted (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) esters. Asused herein, the term (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester” refers toa compound having the following formula:

wherein the C₃₋₈ alkyl group is as defined herein. As used herein theterm “terminal hydroxy-substituted “(C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl)ester” refers to a compound having the following formula:

wherein at least one of the alkyl groups (i.e., at least one of the C₄₋₉alkyl and C₃₋₈ alkyl groups) is subsequently substituted by at least oneterminal hydroxy (—OH) group, and the C₃₋₈ alkyl is as defined herein.As used herein, the term “C₄₋₉ alkyl” refers to a saturated hydrocarbongroup that may be straight-chain or branched, having 4 to 9 carbonatoms. In some embodiments, the alkyl group contains 4 to 9, 4 to 8, 4to 7, 4 to 6, 4 to 5, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 9, 6 to 8, 6to 7, 7 to 9, 7 to 8, or 8 to 9 carbon atoms. Example alkyl moietiesinclude n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl,neo-pentyl, n-hexyl, n-heptyl, n-octyl, and n-nonyl. In someembodiments, one of the alkyl groups is substituted by at least oneterminal hydroxy group. In some embodiments, each of the alkyl groups issubstituted by at least one terminal hydroxy group. In some embodiments,one of the alkyl groups is substituted by one terminal hydroxy group. Insome embodiments, each of the alkyl groups is substituted by oneterminal hydroxy group. In some embodiments, the terminalhydroxy-substituted (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester is selectedfrom the group consisting of:

In some embodiments, the method includes:

a) enzymatically converting C₄₋₉ alkanoyl-CoA to a (C₄₋₉alkyl)-OC(═O)—(C₃₋₈ alkyl) ester; and

b) enzymatically converting the (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) esterto any of (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester, (C₄₋₉hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester, or (C₄₋₉hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester.

In some embodiments, C₄₋₉ alkanoyl-CoA is enzymatically converted to the(C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester using a polypeptide havingalcohol O-acetyltransferase activity. In some embodiments, the alcoholO-acetyltransferase has at least 70% sequence identity to the amino acidsequence set forth in SEQ ID NO: 26.

In some embodiments, the (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester isenzymatically converted to any of (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈hydroxyalkyl) ester, (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl)ester, or (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester using apolypeptide having monooxygenase activity. In some embodiments, thepolypeptide having monooxygenase activity is classified under EC1.14.14.- or EC 1.14.15.-.

In some embodiments, the method further includes enzymaticallyconverting (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester or (C₄₋₉alkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester to a C₄₋₉ hydroxyalkanoate. Insome embodiments, a polypeptide having esterase activity enzymaticallyconverts (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester or (C₄₋₉alkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester to the C₄₋₉ hydroxyalkanoate.

As used herein, the term C₄₋₉ hydroxyalkanoate refers to a compoundhaving the following formula:

wherein the C₃₋₈ hydroxyalkyl is as defined herein. Example C₄₋₉hydroxyalkanoates include, but are not limited to, 5-hydroxypentanoate(i.e., 5-hydroxypentanoic acid), 4-hydroxypentanoate (i.e.,4-hydroxypentanoic acid), 3-hydroxypentanoate (i.e., 3-hydroxypentanoicacid), and the like. It is understood by those skilled in the art thatthe specific form will depend on pH (e.g., neutral or ionized forms,including any salt forms thereof). In some embodiments, the C₃₋₈hydroxyalkyl is a group having the following formula:

In some embodiments, the method of producing one or morehydroxy-substituted (C₃₋₈ alkyl)-OC(═O)—(C₃₋₈ alkyl) esters is a methodof producing one or more pentanoic acid pentyl hydroxyl esters. In someembodiments, the method includes:

a) enzymatically converting pentanoyl-CoA to pentanoic acid pentylester; and

b) enzymatically converting pentanoic acid pentyl ester to any of5-hydroxypentanoic acid pentyl ester, 5-hydroxypentanoic acid5-hydroxypentyl ester, or pentanoic acid 5-hydroxypentyl ester.

In some embodiments, pentanoyl-CoA is enzymatically converted topentanoic acid pentyl ester using a polypeptide having alcoholO-acetyltransferase activity. In some embodiments, the polypeptidehaving alcohol O-acetyltransferase activity has at least 70% sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 26.

In some embodiments, pentanoic acid pentyl ester is enzymaticallyconverted to any of 5-hydroxypentanoic acid pentyl ester,5-hydroxypentanoic acid 5-hydroxypentyl ester and/or pentanoic acid5-hydroxypentyl ester using a polypeptide having monooxygenase activity.In some embodiments, the polypeptide having monooxygenase activity isclassified under EC 1.14.14.- or EC 1.14.15.-.

In some embodiments, the method further includes enzymaticallyconverting 5-hydroxypentanoic acid 5-hydroxypentyl ester or5-hydroxypentanoic acid pentyl ester to 5-hydroxypentanoate. In someembodiments, a polypeptide having esterase activity enzymaticallyconverts 5-hydroxypentanoic acid 5-hydroxypentyl ester or5-hydroxypentanoic acid pentyl ester to 5-hydroxypentanoate.

In some embodiments, the method can include enzymatically converting5-hydroxypentanoic acid 5-hydroxypentyl ester or pentanoic acid5-hydroxypentyl ester to 1,5-pentanediol. In some embodiments, apolypeptide having esterase activity enzymatically converts5-hydroxypentanoic acid 5-hydroxypentyl ester or pentanoic acid5-hydroxypentyl ester to 1,5-pentanediol.

In some embodiments, the method further includes enzymaticallyconverting 5-hydroxypentanoic acid pentyl ester, 5-hydroxypentanoic acid5-hydroxypentyl ester, or pentanoic acid 5-hydroxypentyl ester to5-hydroxypentanoate and/or 1,5-pentanediol. In some embodiments, apolypeptide having esterase activity enzymatically converts5-hydroxypentanoic acid pentyl ester, 5-hydroxypentanoic acid5-hydroxypentyl ester, or pentanoic acid 5-hydroxypentyl ester to5-hydroxypentanoate and/or 1,5-pentanediol.

In some embodiments, the method further includes enzymaticallyconverting 1,5-pentanediol to 5-hydroxypentanal. In some embodiments, apolypeptide having alcohol dehydrogenase activity enzymatically converts1,5-pentanediol to 5-hydroxypentanal.

In some embodiments, the method further includes enzymaticallyconverting 5-hydroxypentanal to 5-hydroxypentanoate. In someembodiments, a polypeptide having aldehyde dehydrogenase activityenzymatically converts 5-hydroxypentanal to 5-hydroxypentanoate.

In some embodiments, the method further includes enzymaticallyconverting 5-hydroxypentanoate methyl ester to 5-hydroxypentanoate. Insome embodiments, a polypeptide having demethylase or esterase activityenzymatically converts 5-hydroxypentanoate methyl ester to5-hydroxypentanoate.

In some embodiments, the method further includes enzymaticallyconverting 5-hydroxypentanoate to a product selected from the groupconsisting of glutaric acid, glutarate semialdehyde, 5-aminopentanoate,cadaverine, and 1,5-pentanediol.

In some embodiments, the method includes enzymatically converting5-hydroxypentanoate to glutarate semialdehyde using a polypeptide havingalcohol dehydrogenase activity, a polypeptide having 6-hydroxyhexanoatedehydrogenase activity, a polypeptide having 5-hydroxypentanoatedehydrogenase activity, a polypeptide having 4-hydroxybutyratedehydrogenase activity, or a polypeptide having monooxygenase activity.

In some embodiments, the method further includes enzymaticallyconverting glutarate semialdehyde to glutaric acid using a polypeptidehaving 7-oxoheptanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having5-oxopentanoate dehydrogenase activity, a polypeptide having aldehydedehydrogenase activity, or a polypeptide having monooxygenase activity.

In some embodiments, the method further includes enzymaticallyconverting glutarate semialdehyde to 5-aminopentanoate. In someembodiments, a ω-transaminase enzymatically converts glutaratesemialdehyde to 5-aminopentanoate.

In some embodiments, the method further includes enzymaticallyconverting 5-aminopentanoate to cadaverine. In some embodiments, themethod further includes enzymatically converting glutarate semialdehydeto cadaverine. In some embodiments, glutarate semialdehyde or5-aminopentanoate is enzymatically converted to cadaverine using apolypeptide having carboxylate reductase activity and a polypeptidehaving ω-transaminase activity and optionally one or more of apolypeptide having N-acetyl transferase activity, a polypeptide havingacetylputrescine deacetylase activity, and a polypeptide having alcoholdehydrogenase activity.

In some embodiments, 5-hydroxypentanoate is enzymatically converted to1,5-pentanediol using a polypeptide having carboxylate reductaseactivity and a polypeptide having alcohol dehydrogenase activity.

In some embodiments, said method further comprises enzymaticallyconverting 1,5-pentanediol to cadaverine. In some embodiments, apolypeptide having alcohol dehydrogenase activity and a polypeptidehaving ω-transaminase activity enzymatically converts 1,5-pentanediolcadaverine.

In some embodiments, a polypeptide having carboxylate reductaseactivity, a polypeptide having ω-transaminase activity, and apolypeptide having alcohol dehydrogenase activity enzymatically converts5-hydroxypentanoate to cadaverine. In some embodiments, theω-transaminase has at least 70% sequence identity to any one of theamino acid sequences set forth in SEQ ID NO. 8-13.

In some embodiments, pentanoyl-CoA is produced from acetyl-CoA andpropanoyl-CoA via two cycles of CoA-dependent carbon chain elongation.In some embodiments, each of said two cycles of CoA-dependent carbonchain elongation comprises using a polypeptide having β-ketothiolaseactivity or a polypeptide having acetyl-CoA carboxylase activity and apolypeptide having β-ketoacyl-[acp] synthase activity, a polypeptidehaving 3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity, and a polypeptide having trans-2-enoyl-CoA reductaseactivity to form pentanoyl-CoA from acetyl-CoA and propanoyl-CoA.

Enzymes Converting Pentanoate or Pentanoyl-CoA to 5-Hydroxypentanoate

As depicted in FIG. 7, pentanoate methyl ester can be formed frompentanoate using a polypeptide having fatty acid O-methyltransferaseactivity, such as the fatty acid O-methyltransferase classified, forexample, under EC 2.1.1.15. For example, the polypeptide having fattyacid O-methyltransferase activity can be obtained from Mycobacteriummarinum (GenBank Accession No. ACC41782.1. SEQ ID NO:23); Mycobacteriumsmegmatis (see GenBank Accession No. ABK73223.1, SEQ ID NO: 24), orPseudomonas putida (see GenBank Accession No. CAA39234.1, SEQ ID NO:25).

Pentanoate methyl ester can be converted to 5-hydroxypentanoate methylester using a polypeptide having monooxygenase activity classified, forexample, under EC 1.14.14.- or EC 1.14.15.- (e.g., EC 1.14.15.1 or EC1.14.15.3) For example, a polypeptide having monooxygenase activity canbe, for example, from the CYP153A family (SEQ ID NOs:14-16), the CYP52A3family (See Genbank Accession No: BAA00371.1, SEQ ID NO: 29) or the alkBfamily such as the gene product of alkBGT from Pseudomonas putida (SeeGenbank Accession No. CAB51047.1, SEQ ID NO: 28). See, FIG. 7.

5-hydroxypentanoate methyl ester can be converted to 5-hydroxypentanoateusing a polypeptide having demethylase activity classified, for example,under EC 2.1.1.- such as the gene product of ligM (see GenBank AccessionNo. BAD61059.1; SEQ ID NO:31) or desA (GenBank Accession No. BAC59257.1;SEQ ID NO:32) or using a polypeptide having esterase activityclassified, for example under EC 3.1.1.- such as the gene product ofEstC (see GenBank Accession No. AAC60471.2, SEQ ID NO: 27).

As depicted in FIG. 7, pentanoyl-CoA can be converted to pentanoic acidpentyl ester using a polypeptide having alcohol O-acetyltransferaseactivity classified, for example, under EC 2.3.1.- (e.g., EC 2.3.1.84)such as the gene product of Eht1 (Genbank Accession No: CAA85138.1, SEQID NO: 26).

Pentanoic acid pentyl ester can be converted to 5-hydroxypentanoic acidpentyl ester and/or 5-hydroxypentanoic acid 5-hydroxypentyl ester usinga polypeptide having monooxygenase activity classified, for example,under EC 1.14.14.- or EC 1.14.15.- (e.g., EC 1.14.15.1 or EC 1.14.15.3)For example, a polypeptide having monooxygenase activity can be, forexample, from the CYP153A family, the CYP52A3 family (Genbank AccessionNo: BAA00371.1, SEQ ID NO: 29) or the alkB family such as the geneproduct of alkBGT from Pseudomonas putida (Genbank Accession No.CAB51047.1, SEQ ID NO: 28). See, FIG. 7.

5-hydroxypentanoic acid pentyl ester and 5-hydroxypentanoic acid5-hydroxypentyl can be converted to 5-hydroxypentanoate using apolypeptide having esterase activity classified, for example, under EC3.1.1.- (EC 3.1.1.1 or EC 3.1.1.6) such as the gene product of EstC (seeGenBank Accession No. AAC60471.2, SEQ ID NO: 27).

For example, the monooxygenase CYP153A family classified, for example,under EC 1.14.15.- (e.g., EC 1.14.15.1 or EC 1.14.15.3) is soluble andhas regio-specificity for terminal hydroxylation, accepting medium chainlength substrates (see, e.g., Koch et al., Appl. Environ. Microbiol.,2009, 75(2), 337-344; Funhoff et al., 2006, J. Bacteriol., 188(44):5220-5227; Van Beilen & Funhoff, Current Opinion in Biotechnology, 2005,16, 308-314; Nieder and Shapiro, J. Bacteriol., 1975, 122(1), 93-98).Although non-terminal hydroxylation is observed in vitro for CYP153A, invivo only 1-hydroxylation occurs (see, Funhoff et al., 2006, supra).

The substrate specificity and activity of terminal monooxygenases hasbeen broadened via successfully, reducing the chain length specificityof CYP153A to below C8 (Koch et al., 2009, supra).

In some embodiments, pentanoate can be enzymatically formed frompentanoyl-CoA using a polypeptide having thioesterase activityclassified under EC 3.1.2.-, such as the gene product of YciA, tesB(GenBank Accession No. AAA24665.1, SEQ ID NO: 1) or Acot13 (Cantu etal., Protein Science, 2010, 19, 1281-1295; Zhuang et al., Biochemistry,2008, 47(9):2789-2796; Naggert et al., J. Biol. Chem., 1991, 266(17):11044-11050), the acyl-[acp] thioesterase from a Lactobacillus brevis(GenBank Accession No. ABJ63754.1, SEQ ID NO:33), or a Lactobacillusplantarum (GenBank Accession No. CCC58182.1, SEQ ID NO:34). Suchacyl-[acp] thioesterases have C6-C8 chain length specificity (see, forexample, Jing et al., 2011, BMC Biochemistry, 12(44)). See, FIG. 3.

In some embodiments, pentanoate can be enzymatically formed frompentanoyl-CoA using a polypeptide having butanal dehydrogenase activityclassified, for example, under EC 1.2.1.- such as EC 1.2.1.10 or EC1.2.1.57 (see, e.g., GenBank Accession No. AAD39015, SEQ ID NO:30)(e.g., the gene product of PduB or PduD) or an aldehyde dehydrogenaseclassified, for example, under EC 1.2.1.- such as EC 1.2.1.3 or EC1.2.1.4 (see, Ho & Weiner, J. Bacteriol., 2005, 187(3):1067-1073). See,FIG. 3.

Enzymes Generating Pentanoyl-CoA for Conversion to a C5 Building Block

As depicted in FIG. 1 and FIG. 2, pentanoyl-CoA can be formed fromacetyl-CoA or propanoyl-CoA via two cycles of CoA-dependent carbon chainelongation using either NADH or NADPH dependent enzymes.

In some embodiments, a CoA-dependent carbon chain elongation cyclecomprises using a polypeptide having β-ketothiolase activity or apolypeptide having acetyl-CoA carboxylase activity and a polypeptidehaving β-ketoacyl-[acp] synthase activity, a polypeptide having3-hydroxyacyl-CoA dehydrogenase activity or a polypeptide having3-oxoacyl-CoA reductase activity, a polypeptide having enoyl-CoAhydratase activity and a polypeptide having trans-2-enoyl-CoA reductaseactivity. A polypeptide having β-ketothiolase activity can convertpropanoyl-CoA to 3-oxopentanoyl-CoA and can convert pentanoyl-CoA to3-oxopentanoyl-CoA. A polypeptide having acetyl-CoA carboxylase activitycan convert acetyl-CoA to malonyl-CoA. A polypeptide havingacetoacetyl-CoA synthase activity can convert malonyl-CoA toacetoacetyl-CoA. A polypeptide having 3-hydroxybutyryl-CoA dehydrogenaseactivity can convert 3-oxopentanoyl-CoA to 3-hydroxypentanoyl CoA. Apolypeptide having 3-oxoacyl-CoA reductase/3-hydroxyacyl-CoAdehydrogenase activity can convert 3-oxopentanoyl-CoA to3-hydroxypentanoyl-CoA. A polypeptide having enoyl-CoA hydrataseactivity can convert 3-hydroxypentanoyl-CoA to pent-2-enoyl-CoA and canconvert 3-hydroxypentanoyl-CoA to pent-2-enoyl-CoA. A polypeptide havingtrans-2-enoyl-CoA reductase activity can convert pent-2-enoyl-CoA topentanoyl-CoA and can convert pent-2-enoyl-CoA to pentanoyl-CoA. SeeFIGS. 1 and 2.

In some embodiments, a polypeptide having β-ketothiolase activity can beclassified under EC 2.3.1.16, such as the gene product of bktB (See,e.g., Genbank Accession AAC38322.1). The polypeptide havingβ-ketothiolase activity encoded by bktB from Cupriavidus necator acceptspropanoyl-CoA and pentanoyl-CoA as substrates. When pentanoyl-CoA is thesubstrate, the CoA-activated C5 aliphatic backbone (3-oxopentanoyl-CoA)is produced (see, e.g., Haywood et al., FEMS Microbiology Letters, 1988,52:91-96; Slater et al., J. Bacteriol., 1998, 180(8):1979-1987). Thepolypeptide having β-ketothiolase activity encoded by paaJ (See, e.g.,Genbank Accession No. AAC54479.1), catF and pcaF can be classifiedunder, for example, EC 2.3.1.174. The polypeptide having β-ketothiolaseactivity encoded by paaJ condenses acetyl-CoA and succinyl-CoA to3-oxoadipyl-CoA (see, for example, Fuchs et al., 2011, Nature ReviewsMicrobiology, 9, 803-816; Gobel et al., 2002, J. Bacteriol., 184(1),216-223) See FIGS. 1 and 2.

In some embodiments, a polypeptide having acetyl-CoA carboxylaseactivity can be classified, for example, under EC 6.4.1.2. In someembodiments, a polypeptide having β-ketoacyl-[acp] synthase activity canbe classified, for example, under 2.3.1.180 such as the gene product ofFabH from Staphylococcus aereus (Qiu et al., 2005, Protein Science, 14:2087-2094). See FIGS. 1 and 2.

In some embodiments, a polypeptide having 3-hydroxyacyl-CoAdehydrogenase activity or a polypeptide having 3-oxoacyl-CoAdehydrogenase activity can be classified under EC 1.1.1.-. For example,the polypeptide having 3-hydroxyacyl-CoA dehydrogenase activity can beclassified under EC 1.1.1.35, such as the gene product of fadB (FIG. 1);classified under EC 1.1.1.157, such as the gene product of hbd (can bereferred to as a 3-hydroxybutyryl-CoA dehydrogenase) (FIG. 1); orclassified under EC 1.1.1.36, such as the acetoacetyl-CoA reductase geneproduct of phaB (Liu & Chen, Appl. Microbiol. Biotechnol., 2007,76(5):1153-1159; Shen et al., Appl. Environ. Microbiol., 2011,77(9):2905-2915; Budde et al., J. Bacteriol., 2010, 192(20):5319-5328)(FIG. 2).

In some embodiments, a polypeptide having 3-oxoacyl-CoA reductaseactivity can be classified under EC 1.1.1.100, such as the gene productof fabG (Budde et al., J. Bacteriol., 2010, 192(20):5319-5328; Nomura etal., Appl. Environ. Microbiol., 2005, 71(8):4297-4306). FIG. 2.

In some embodiments, a polypeptide having enoyl-CoA hydratase activitycan be classified under EC 4.2.1.17, such as the gene product of crt(Genbank Accession No. AAA95967.1) (FIG. 1), or classified under EC4.2.1.119, such as the gene product of phaJ (Genbank Accession No.BAA21816.1) (FIG. 2) (Shen et al., 2011, supra; Fukui et al., J.Bacteriol., 1998, 180(3):667-673).

In some embodiments, a polypeptide having trans-2-enoyl-CoA reductaseactivity can be classified under EC 1.3.1.38 (FIG. 2), EC 1.3.1.8 (FIG.2), or EC 1.3.1.44 (FIG. 1), such as the gene product of ter (GenbankAccession No. AAW66853.1) (Nishimaki et al., J. Biochem., 1984,95:1315-1321; Shen et al., 2011, supra) or tdter (Genbank Accession No.AAS11092.1) (Bond-Watts et al., Biochemistry, 2012, 51:6827-6837).

Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis of aC5 Building Block

As depicted in FIGS. 4, 5, and 7, a terminal carboxyl group can beenzymatically formed using a polypeptide having thioesterase activity, apolypeptide having aldehyde dehydrogenase activity, a polypeptide having7-oxoheptanoate dehydrogenase activity, a polypeptide having6-oxohexanoate dehydrogenase activity, a polypeptide having5-oxopentanoate dehydrogenase activity, a polypeptide havingmonooxygenase activity, a polypeptide having esterase activity, or apolypeptide having demethylase activity.

In some embodiments, the first terminal carboxyl group is enzymaticallyformed by a polypeptide having syringate O-demethylase activityclassified under EC 2.1.1.- such as the gene products of ligM (seeGenBank Accession No. BAD61059.1; SEQ ID NO:31) or desA (GenBankAccession No. BAC59257.1; SEQ ID NO:32) or an esterase classified underEC 3.1.1.- such as the gene product of EstC (see, e.g., GenBankAccession No. AAC60471.2, SEQ ID NO: 27) See, e.g., FIG. 7.

In some embodiments, the first terminal carboxyl group is enzymaticallyformed by a polypeptide having aldehyde dehydrogenase activityclassified, for example, under EC 1.2.1.3 or EC 1.2.1.4.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of glutaric acid is enzymatically formed by a polypeptidehaving aldehyde dehydrogenase activity classified, for example, under EC1.2.1.3 (see, Guerrillot & Vandecasteele, Eur. J. Biochem., 1977, 81,185-192). See FIG. 4.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of glutaric acid is enzymatically formed by a polypeptidehaving dehydrogenase activity classified under EC 1.2.1.- such as a5-oxopentanoate dehydrogenase (e.g., the gene product of CpnE), a6-oxohexanoate dehydrogenase (e.g., the gene product of ChnE fromAcinetobacter sp.), a 7-oxoheptanoate dehydrogenase (e.g., the geneproduct of ThnG from Sphingomonas macrogolitabida) (Iwaki et al., Appl.Environ. Microbiol., 1999, 65(11), 5158-5162; López-Sánchez et al.,Appl. Environ. Microbiol., 2010, 76(1), 110-118). For example, apolypeptide having 5-oxopentanoate dehydrogenase activity can beclassified under EC 1.2.1.20. For example, a polypeptide having6-oxohexanoate dehydrogenase activity can be classified under EC1.2.1.63. For example, a polypeptide having 7-oxoheptanoatedehydrogenase activity can be classified under EC 1.2.1.-. See, FIG. 4.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of glutaric acid is enzymatically formed by a polypeptidehaving monooxygenase activity in the cytochrome P450 family such asCYP4F3B (see, e.g., Sanders et al., J. Lipid Research, 2005,46(5):1001-1008; Sanders et al., The FASEB Journal, 2008,22(6):2064-2071). See, FIG. 4.

The utility of ω-oxidation in introducing carboxyl groups into alkaneshas been demonstrated in the yeast Candida tropicalis, leading to thesynthesis of adipic acid (Okuhara et al., Agr. Biol. Chem., 1971, 35(9),1376-1380).

Enzymes Generating the Terminal Amine Groups in the Biosynthesis of a C5Building Block

As depicted in FIG. 5 and FIG. 6, terminal amine groups can beenzymatically formed using a polypeptide having ω-transaminase activityor a polypeptide having deacetylase activity.

In some embodiments, the first terminal amine group leading to thesynthesis of 5-aminopentanoic acid, 5-aminopentanal, or 5-aminopentanolis enzymatically formed by a ω-transaminase classified, for example,under EC 2.6.1.- such as EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC2.6.1.48, or EC 2.6.1.82 such as that obtained from Chromobacteriumviolaceum (Genbank Accession No. AAQ59697.1, SEQ ID NO: 8), Pseudomonasaeruginosa (Genbank Accession No. AAG08191.1, SEQ ID NO: 9), Pseudomonassyringae (Genbank Accession No. AAY39893.1, SEQ ID NO: 10), Rhodobactersphaeroides (Genbank Accession No. ABA81135.1, SEQ ID NO: 11), Vibriofluvialis (Genbank Accession No. AAA57874.1, SEQ ID NO: 13),Streptomyces griseus, or Clostridium viride. An additional polypeptidehaving ω-transaminase activity that can be used in the methods and hostsdescribed herein is from Escherichia coli (Genbank Accession No.AAA57874.1, SEQ ID NO: 12). Some of the polypeptides havingω-transaminases activity classified, for example, under EC 2.6.1.29 orEC 2.6.1.82 are polypeptides having diamine ω-transaminases activity(e.g., SEQ ID NO:12). See, e.g., FIGS. 5 and 6.

The reversible polypeptide having ω-transaminase activity fromChromobacterium violaceum (Genbank Accession No. AAQ59697.1, SEQ ID NO:8) has demonstrated analogous activity accepting 6-aminohexanoic acid asamino donor, thus forming the first terminal amine group in adipatesemialdehyde (Kaulmann et al., Enzyme and Microbial Technology, 2007,41, 628-637).

The reversible polypeptide having 4-aminobutyrate:2-oxoglutaratetransaminase activity from Streptomyces griseus has demonstratedanalogous activity for the conversion of 6-aminohexanoate to adipatesemialdehyde (Yonaha et al., Eur. J. Biochem., 1985, 146, 101-106).

The reversible polypeptide having 5-aminovalerate transaminase activityfrom Clostridium viride has demonstrated analogous activity for theconversion of 6-aminohexanoate to adipate semialdehyde (Barker et al.,J. Biol. Chem., 1987, 262(19), 8994-9003).

In some embodiments, the second terminal amine group leading to thesynthesis of cadaverine is enzymatically formed by a polypeptide havingdiamine transaminase activity. For example, the second terminal aminogroup can be enzymatically formed by a polypeptide having diaminetransaminase activity classified, for example, under EC 2.6.1.-, e.g.,EC 2.6.1.29 or classified, for example, under EC 2.6.1.82, such as thegene product of YgjG from E. coli (Genbank Accession No. AAA57874.1, SEQID NO: 12). See, FIG. 6.

The gene product of ygjG accepts a broad range of diamine carbon chainlength substrates, such as putrescine, cadaverine and spermidine(Samsonova et al., BMC Microbiology, 2003, 3:2).

The polypeptide having diamine transaminase activity from E. coli strainB has demonstrated activity for 1,7 diaminopentane (Kim, The Journal ofChemistry, 1964, 239(3), 783-786).

In some embodiments, the second terminal amine group leading to thesynthesis of cadaverine is enzymatically formed by a polypeptide havingdeacetylase activity classified, for example, under EC 3.5.1.62 such asa polypeptide having acetylputrescine deacetylase activity. Thepolypeptide having acetylputrescine deacetylase activity fromMicrococcus luteus K-11 accepts a broad range of carbon chain lengthsubstrates, such as acetylputrescine, acetylcadaverine andN⁸-acetylspermidine (see, for example, Suzuki et al., 1986, BBA—GeneralSubjects, 882(1):140-142).

Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis of aC5 Building Block

As depicted in FIG. 8, a terminal hydroxyl group can be enzymaticallyforming using a polypeptide having alcohol dehydrogenase activity. Forexample, the second terminal hydroxyl group leading to the synthesis of1,7 pentanediol is enzymatically formed by a polypeptide having alcoholdehydrogenase activity classified under EC 1.1.1.- (e.g., EC 1.1.1.1,1.1.1.2, 1.1.1.21, or 1.1.1.184).

A first terminal hydroxyl group can be enzymatically formed with apolypeptide having monooxygenase activity as discussed above withrespect to the formation of 5-hydroxypentanoate methyl ester in FIG. 7.

As depicted in FIG. 8, pentanoyl-CoA can be converted to pentanoic acidpentyl ester using a polypeptide having alcohol O-acetyltransferaseactivity classified, for example, under EC 2.3.1.-(84) such as the geneproduct of Eht1 (Genbank Accession No: CAA85138.1, SEQ ID NO: 26).

Pentanoic acid pentyl ester can be converted to 5-hydroxypentanoic acid5-hydroxypentyl ester using a polypeptide having monooxygenase activityclassified, for example, under EC 1.14.14.- or EC 1.14.15.- (EC1.14.15.1 or EC 1.14.15.3). Pentanoic acid pentyl ester can be convertedto pentanoic acid 5-hydroxypentyl ester using a polypeptide havingmonooxygenase activity classified, for example, under EC 1.14.14.- or EC1.14.15.- (1,3). For example, a polypeptide having monooxygenaseactivity can be, for example, from the CYP153A family, the CYP52A3family or the alkB family such as the gene product of alkBGT fromPseudomonas putida. See, FIG. 7.

Pentanoic acid 5-hydroxypentyl ester and 5-hydroxypentanoic acid5-hydroxypentyl can be converted to 1,5-pentanediol using a polypeptidehaving esterase activity classified, for example, under EC 3.1.1.-(e.g., EC 3.1.1.1 or EC 3.1.1.6) such as the gene product of EstC (seeGenBank Accession No. AAC60471.2, SEQ ID NO: 27).

Biochemical Pathways

Pathways to Propanoyl-CoA

In some embodiments, propanoyl-Coenzyme A (CoA) is a precursor leadingto one or more central precursors in the synthesis of one or more C5building blocks (see, e.g., FIG. 9).

In some embodiments, propanoyl-CoA is synthesized from the centralmetabolite succinyl-CoA by conversion of succinyl-CoA to(2R)-methylmalonyl-CoA by a polypeptide having methylmalonyl-CoA mutaseactivity classified, for example, under EC 5.4.99.2; followed byconversion to (2S)-methylmalonyl-CoA by a polypeptide havingmethylmalonyl-CoA epimerase activity classified, for example, under EC5.1.99.1; followed by conversion to propanoyl-CoA by a polypeptidehaving methylmalonyl-CoA carboxytransferase activity classified, forexample, under EC 2.1.3.1 or a polypeptide having methylmalonyl-CoAdecarboxylase activity classified, for example, under EC 4.1.1.41. Seee.g., FIG. 9.

In some embodiments, propanoyl-CoA is synthesized from the centralmetabolite, L-threonine, by conversion of L-threonine to 2-oxobutyrateby a polypeptide having threonine ammonia lyase activity classified, forexample, under EC 4.3.1.19; followed by conversion to propanoyl-CoA by apolypeptide having 2-ketobutyrate formate-lyase activity classified, forexample, under EC 2.3.1.- such as the gene product of tdcE (Tseng etal., Microbial Cell Factories, 2010, 9:96). See, e.g., FIG. 9.

In some embodiments, propanoyl-CoA is synthesized from 1,2-propanediolby conversion to propanal by a polypeptide having propanedioldehydratase activity classified, for example, under EC 4.2.1.28;followed by conversion to propanoyl-CoA by a polypeptide havingCoA-dependent propionaldehyde dehydrogenase activity such as the geneproduct of pduP (Luo et al., Bioresource Technology, 2012, 103:1-6).See, e.g., FIG. 9.

In some embodiments, propanoyl-CoA is synthesized from the carbonsource, levulinic acid, by conversion of levulinic acid to levulinyl-CoAby a polypeptide having acyl-CoA synthetase or ligase activityclassified, for example, under EC 6.2.1.-; followed by conversion topropanoyl-CoA by a transferase classified, for example, under EC 2.3.1.-(Jaremko and Yu, J. Biotechnol., 2011, 155:293-298). See, e.g., FIG. 9.

In some embodiments, propanoyl-CoA is synthesized from the centralmetabolite, pyruvate, by conversion of pyruvate to L-lactate by apolypeptide having L-lactate dehydrogenase activity classified, forexample, under EC 1.1.1.27; followed by conversion to lactoyl-CoA by apolypeptide having proprionate CoA-transferase activity classified, forexample, under EC 2.8.3.1; followed by conversion to propenoyl-CoA by apolypeptide having lactoyl-CoA dehydratase activity classified, forexample, under EC 4.2.1.54; followed by conversion to propanoyl-CoA by apolypeptide having butyryl-CoA dehydrogenase activity classified, forexample, under EC 1.3.8.1 or a polypeptide having medium-chain acyl-CoAdehydrogenase activity classified, for example, under EC 1.3.8.7. See,e.g., FIG. 9.

In some embodiments, propanoyl-CoA is synthesized from the centralmetabolite, malonyl-CoA, by conversion of malonyl-CoA to malonatesemialdehyde by a polypeptide having malonyl-CoA reductase activityclassified, for example, under EC 1.2.1.75; followed by conversion to3-hydroxypropionate by a polypeptide having 3-hydroxypropionatedehydrogenase activity classified, for example, under EC 1.1.1.59;followed by conversion to 3-hydroxypropionyl-CoA by a polypeptide having3-hydroxyisobutyryl-CoA hydrolase activity classified, for example,under EC 6.2.1.- such as EC 6.2.1.36; followed by conversion topropenoyl-CoA by a polypeptide having 3-hydroxypropionyl-CoA dehydrataseactivity classified, for example, under EC 4.2.1.116; followed byconversion to propanoyl-CoA by a polypeptide having butyryl-CoAdehydrogenase activity classified, for example, under EC 1.3.8.1 or apolypeptide having medium-chain acyl-CoA dehydrogenase activityclassified, for example, under EC 1.3.8.7. See, e.g., FIG. 9.

Pathways to Pentanoyl-CoA as Central Precursor to C5 Building Blocks

In some embodiments, pentanoyl-CoA is synthesized from propanoyl-CoA byconversion of propanoyl-CoA to 3-oxopentanoyl-CoA by a polypeptidehaving β-ketothiolase activity classified, for example, under EC2.3.1.16, such as the gene product of bktB (Genbank Accession No.AAC38322.1) or classified, for example, under EC 2.3.1.174 such as thegene product of paaJ (Genbank Accession No. AAC54479.1); followed byconversion of 3-oxopentanoyl-CoA to (S) 3-hydroxybutanoyl-CoA by apolypeptide having 3-hydroxyacyl-CoA dehydrogenase activity classified,for example, under EC 1.1.1.35, such as the gene product of fadB orclassified, for example, under EC 1.1.1.157 such as the gene product ofhbd; followed by conversion of (S) 3-hydroxypentanoyl-CoA topent-2-enoyl-CoA by a polypeptide having enoyl-CoA hydratase activityclassified, for example, under EC 4.2.1.17 such as the gene product ofcrt (Genbank Accession No. AAA95967.1); followed by conversion ofpent-2-enoyl-CoA to pentanoyl-CoA by a polypeptide havingtrans-2-enoyl-CoA reductase activity classified, for example, under EC1.3.1.44 such as the gene product of ter (Genbank Accession No.AAW66853.1) or tdter (Genbank Accession No. AAS11092.1); followed byconversion of pentanoyl-CoA to 3-oxo-pentanoyl-CoA by a polypeptidehaving β-ketothiolase activity classified, for example, under EC2.3.1.16 such as the gene product of bktB (Genbank Accession No.AAC38322.1) or classified, for example, under EC 2.3.1.174 such as thegene product of paaJ (Genbank Accession No. AAC54479.1); followed byconversion of 3-oxo-pentanoyl-CoA to (S) 3-hydroxypentanoyl-CoA by apolypeptide having 3-hydroxyacyl-CoA dehydrogenase activity classified,for example, under EC 1.1.1.35 such as the gene product of fadB or by apolypeptide having 3-hydroxyacyl-CoA dehydrogenase activity classified,for example, under EC 1.1.1.157 such as the gene product of hbd;followed by conversion of (S) 3-hydroxypentanoyl-CoA to pent-2-enoyl-CoAby a polypeptide having enoyl-CoA hydratase activity classified, forexample, under EC 4.2.1.17 such as the gene product of crt (GenbankAccession No. AAA95967.1); followed by conversion of pent-2-enoyl-CoA topentanoyl-CoA by a polypeptide having trans-2-enoyl-CoA reductaseactivity classified, for example, under EC 1.3.1.44 such as the geneproduct of ter (Genbank Accession No. AAW66853.1) or tdter (GenbankAccession No. AAS11092.1). See FIG. 1.

In some embodiments, pentanoyl-CoA is synthesized from the centralmetabolite, propanoyl-CoA, by conversion of propanoyl-CoA to3-oxopentanoyl-CoA by a polypeptide having β-ketothiolase activityclassified, for example, under EC 2.3.1.16, such as the gene product ofbktB; followed by conversion of 3-oxopentanoyl-CoA to (R)3-hydroxypentanoyl-CoA by a polypeptide having 3-oxoacyl-CoA reductaseactivity classified, for example, under EC 1.1.1.100, such as the geneproduct of fadG or by a polypeptide having acetoacetyl-CoA reductaseactivity classified, for example, under EC 1.1.1.36 such as the geneproduct of phaB; followed by conversion of (R) 3-hydroxypentanoyl-CoA topent-2-enoyl-CoA by a polypeptide having enoyl-CoA hydratase activityclassified, for example, under EC 4.2.1.119 such as the gene product ofphaJ (Genbank Accession No. BAA21816.1); followed by conversion ofpent-2enoyl-CoA to pentanoyl-CoA by a polypeptide havingtrans-2-enoyl-CoA reductase activity classified, for example, under EC1.3.1.38 or a polypeptide having acyl-CoA dehydrogenase activityclassified, for example, under EC 1.3.1.8; followed by conversion ofpentanoyl-CoA to 3-oxo-pentanoyl-CoA by a polypeptide havingβ-ketothiolase activity classified, for example, under EC 2.3.1.16 suchas the gene product of bktB (Genbank Accession No. AAC38322.1) orclassified, for example, under EC 2.3.1.174 such as the gene product ofpaaJ (Genbank Accession No. AAC54479.1); followed by conversion of3-oxo-pentanoyl-CoA to (R) 3-hydroxypentanoyl-CoA by a polypeptidehaving 3-oxoacyl-CoA reductase activity classified, for example, underEC 1.1.1.100 such as the gene product of fabG; followed by conversion of(R) 3-hydroxypentanoyl-CoA to pent-2-enoyl-CoA by a polypeptide havingenoyl-CoA hydratase activity classified, for example, under EC 4.2.1.119such as the gene product of phaJ (Genbank Accession No. BAA21816.1);followed by conversion of pent-2-enoyl-CoA to pentanoyl-CoA by apolypeptide having trans-2-enoyl-CoA reductase activity classified, forexample, under EC 1.3.1.38 or a polypeptide having acyl-CoAdehydrogenase activity classified, for example, under EC 1.3.1.8. SeeFIG. 2.

In some embodiments, 3-oxopentanoyl-CoA can be synthesized fromacetyl-CoA. A polypeptide having acetyl-CoA carboxylase activityclassified, for example, under EC 6.4.1.2 can be used to convertacetyl-CoA to malonyl-CoA, which can be converted to 3-oxopentanoyl-CoAusing a polypeptide having β-ketoacyl-[acp] synthase activityclassified, for example, under EC 2.3.1.- such as EC 2.3.1.41, EC2.3.1.179 or EC 2.3.1.180 such as the gene product of fabH. See, FIG. 1and FIG. 2.

Pathways Using Pentanoyl-CoA to Produce the Central Precursor Pentanoate

In some embodiments, pentanoate is synthesized from pentanoyl-CoA byconversion of pentanoyl-CoA to pentanoate by a polypeptide havingthioesterase activity classified, for example, under EC 3.1.2.- such asthe gene product of YciA, tesB, Acot13, a Lactobacillus brevisacyl-[acp] thioesterase (GenBank Accession No. ABJ63754.1, SEQ ID NO:33)or a Lactobacillus plantarum acyl-[acp] thioesterase (GenBank AccessionNo. CCC58182.1, SEQ ID NO:34). See, FIG. 3.

In some embodiments, pentanoyl-CoA is converted to pentanal by apolypeptide having butanal dehydrogenase activity classified, forexample, under EC 1.2.1.- such as EC 1.2.1.10 or EC 1.2.1.57 (see, e.g,GenBank Accession No. AAD39015, SEQ ID NO:30); followed by conversion ofpentanal to pentanoate by a polypeptide having aldehyde dehydrogenaseactivity classified, for example, under EC 1.2.1.4 or EC 1.2.1.3. SeeFIG. 3.

The conversion of hexanoyl-CoA to hexanal has been demonstrated usingboth NADH and NADPH as co-factors (see Palosaari and Rogers, J.Bacteriol., 1988, 170(7): 2971-2976).

Pathways Using Pentanoyl-CoA to Produce the Central Precursor Pentanol

In some embodiments, pentanoate is synthesized from pentanoyl-CoA byconversion of pentanoyl-CoA to pentanoate by a polypeptide havingthioesterase activity classified, for example, under EC 3.1.2.- such asthe gene product of YciA, tesB or Acot13, a Lactobacillus brevisacyl-[acp] thioesterase (GenBank Accession No. ABJ63754.1, SEQ ID NO:33)or a Lactobacillus plantarum acyl-[acp] thioesterase (GenBank AccessionNo. CCC58182.1, SEQ ID NO:34); followed by conversion of pentanoate topentanal by a polypeptide having carboxylate reductase activityclassified, for example, under EC 1.2.99.6, such as the gene product ofcar enhanced by the gene product of sfp; followed by conversion ofpentanal to pentanol by a polypeptide having alcohol dehydrogenaseactivity classified, for example, under EC 1.1.1.- such as EC 1.1.1.1,EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the gene product ofYMR318C (Genbank Accession No. CAA90836.1) or YqhD (from E. coli,GenBank Accession No. AAA69178.1) (see, e.g., Liu et al., Microbiology,2009, 155, 2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1),163-172; or Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257)or the protein having GenBank Accession No. CAA81612.1 (from Geobacillusstearothermophilus). See, FIG. 3.

In some embodiments, pentanoyl-CoA is converted to pentanal by apolypeptide having butanal dehydrogenase activity classified, forexample, under EC 1.2.1.57 (see, e.g., GenBank Accession No. BAD61059.1,SEQ ID NO:31); followed by conversion of pentanal to pentanol by apolypeptide having alcohol dehydrogenase activity classified, forexample, under EC 1.1.1.- such as EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21,or EC 1.1.1.184) such as the gene product of YMR318C (Genbank AccessionNo. CAA90836.1) or YqhD (from E. coli, GenBank Accession No. AAA69178.1)(see, e.g., Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy etal., 2002, Biochem J., 361(Pt 1), 163-172; or Jarboe, 2011, Appl.Microbiol. Biotechnol., 89(2), 249-257) or the protein having GenBankAccession No. CAA81612.1 (from Geobacillus stearothermophilus). See,FIG. 3.

Pathways Using Pentanoate or Pentanoyl-CoA as Central Precursor to5-Hydroxypentanoate

In some embodiments, 5-hydroxypentanoate is synthesized from the centralprecursor, pentanoate, by conversion of pentanoate to pentanoate methylester using a polypeptide having fatty acid O-methyltransferase activityclassified, for example, under EC 2.1.1.15 (e.g., the fatty acidO-methyltransferase from Mycobacterium marinum (GenBank Accession No.ACC41782.1. SEQ ID NO:23), Mycobacterium smegmatis (see GenBankAccession No. ABK73223.1, SEQ ID NO: 24), or Pseudomonas putida (seeGenBank Accession No. CAA39234.1, SEQ ID NO: 25); followed by conversionto 5-hydroxypentanoate methyl ester using a polypeptide havingmonooxygenase activity classified, for example, under EC 1.14.14.- or EC1.14.15.- (e.g., EC 1.14.15.1 or EC 1.14.15.3) such as a monooxygenasein the CYP153A, a CYP52A3 family, or alkB family; followed by conversionto 5-hydroxypentanoate using a polypeptide having syringateO-demethylase activity classified under EC 2.1.1.- such as the geneproducts of ligM (see GenBank Accession No. BAD61059.1; SEQ ID NO:31) ordesA (GenBank Accession No. BAC59257.1; SEQ ID NO:32), or using apolypeptide having esterase activity classified under EC 3.1.1. such asthe gene product of EstC (see GenBank Accession No. AAC60471.2, SEQ IDNO: 27) (Kim et al., 1994, Biosci. Biotech. Biochem, 58(1), 111-116).

In some embodiments, pentanoate can be enzymatically converted to5-hydroxypentanoate by a polypeptide having monooxygenase activity(classified, for example, under EC 1.14.14.- or EC 1.14.15.- such as amonooxygenase in the CYP153A, the CYP52A3 family, and/or the geneproduct of alkB family.

In some embodiments, pentanoyl-CoA can be converted to pentanoic acidpentyl ester using a polypeptide having alcohol O-acetyltransferaseactivity classified, for example, under EC 2.3.1.-(84) such as the geneproduct of Eht1 (Genbank Accession No: CAA85138.1, SEQ ID NO: 25);followed by conversion to 5-hydroxypentanoic acid pentyl ester and/or5-hydroxypentanoic acid 5-hydroxypentyl ester using a polypeptide havingmonooxygenase activity classified, for example, under EC 1.14.14.- or EC1.14.15.- (1,3). For example, a polypeptide having monooxygenaseactivity can be, for example, from the CYP153A family, the CYP52A3family (Genbank Accession No: BAA00371.1, SEQ ID NO: 29) or the alkBfamily such as the gene product of alkBGT from Pseudomonas putida(Genbank Accession No. CAB51047.1, SEQ ID NO: 28); followed byconversion of 5-hydroxypentanoic acid pentyl ester and/or5-hydroxypentanoic acid 5-hydroxypentyl to 5-hydroxypentanoate using apolypeptide having esterase activity classified, for example, under EC3.1.1.-(1,6) such as the gene product of EstC (see GenBank Accession No.AAC60471.2, SEQ ID NO: 27) (Kim et al., 1994, Biosci. Biotech. Biochem,58(1), 111-116). See FIG. 7.

Pathways Using 5-Hydroxypentanoate as Central Precursor to Glutarate

Glutarate semialdehyde can be synthesized by enzymatically converting5-hydroxypentanoate to glutarate semialdehyde using a polypeptide havingalcohol dehydrogenase activity classified, for example, under EC 1.1.1.-such as the gene product of YMR318C (classified, for example, under EC1.1.1.2, see Genbank Accession No. CAA90836.1) (Larroy et al., 2002,Biochem J., 361(Pt 1), 163-172), cpnD (Iwaki et al., 2002, Appl.Environ. Microbiol., 68(11):5671-5684) or gabD (Lütke-Eversloh &Steinbüchel, 1999, FEMS Microbiology Letters, 181(1):63-71), apolypeptide having 6-hydroxyhexanoate dehydrogenase activity classified,for example, under EC 1.1.1.258 such as the gene product of ChnD (Iwakiet al., Appl. Environ. Microbiol., 1999, 65(11):5158-5162), or apolypeptide having cytochrome P450 activity (Sanders et al., J. LipidResearch, 2005, 46(5), 1001-1008; Sanders et al., The FASEB Journal,2008, 22(6), 2064-2071). See, FIG. 4. The polypeptide having alcoholdehydrogenase activity encoded by YMR318C has broad substratespecificity, including the oxidation of C5 alcohols.

Glutarate semialdehyde can be enzymatically converted to glutaric acidusing a polypeptide having aldehyde dehydrogenase activity classified,for example, under EC 1.2.1.- (3,16,20,63,79) such as a polypeptidehaving 7-oxoheptanoate dehydrogenase activity (e.g., the gene product ofThnG), a polypeptide having 6-oxohexanoate dehydrogenase activity (e.g.,the gene product of ChnE), or a polypeptide having aldehydedehydrogenase activity classified under EC 1.2.1.3. See FIG. 4.

Pathway Using 5-Hydroxypentanoate as Central Precursor to5-Aminopentanoate

In some embodiments, 5-aminopentanoate is synthesized from5-hydroxypentanoate by conversion of 5-hydroxypentanoate to glutaratesemialdehyde using a polypeptide having alcohol dehydrogenase activityclassified, for example, under EC 1.1.1.- such as the gene product ofYMR318C (classified, for example, under EC 1.1.1.2, see GenbankAccession No. CAA90836.1) (Larroy et al., 2002, Biochem J., 361(Pt 1),163-172), cpnD (Iwaki et al., 2002, Appl. Environ. Microbiol.,68(11):5671-5684), or gabD (Lütke-Eversloh & Steinbüchel, 1999, FEMSMicrobiology Letters, 181(1):63-71), or a polypeptide having6-hydroxyhexanoate dehydrogenase activity classified, for example, underEC 1.1.1.258 such as the gene product of ChnD (Iwaki et al., Appl.Environ. Microbiol., 1999, 65(11):5158-5162); followed by conversion to5-aminopentanoate by a polypeptide having ω-transaminase activityclassified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC2.6.1.48, EC 2.6.1.82 such as from a Chromobacterium violaceum (seeGenbank Accession No. AAQ59697.1, SEQ ID NO: 7), a Pseudomonas syringae(see Genbank Accession No. AAY39893.1, SEQ ID NO: 9). See FIG. 5.

Pathway Using 5-Aminopentanoate, 5-Hydroxypentanoate, or GlutarateSemialdehyde as Central Precursor to Cadaverine

In some embodiments, cadaverine is synthesized from the centralprecursor 5-aminopentanoate by conversion of 5-aminopentanoate to5-aminopentanal by a polypeptide having carboxylate reductase activityclassified, for example, under EC 1.2.99.6 such as the gene product ofcar in combination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia)or the gene products of GriC and GriD from Streptomyces griseus;followed by conversion of 5-aminopentanal to cadaverine by a polypeptidehaving ω-transaminase activity classified, for example, under EC 2.6.1.-such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82such as from a Chromobacterium violaceum (see Genbank Accession No.AAQ59697.1, SEQ ID NO: 8), a Pseudomonas aeruginosa (see GenbankAccession No. AAG08191.1, SEQ ID NO: 9), a Pseudomonas syringae (seeGenbank Accession No. AAY39893.1, SEQ ID NO: 10), or an Escherichia coli(see Genbank Accession No. AAA57874.1, SEQ ID NO: 12). See FIG. 6.

The polypeptide having carboxylate reductase activity encoded by thegene product of car and enhancer npt or sfp has broad substratespecificity, including terminal difunctional C4 and C5 carboxylic acids(Venkitasubramanian et al., Enzyme and Microbial Technology, 2008, 42,130-137).

In some embodiments, cadaverine is synthesized from the centralprecursor 5-hydroxypentanoate (which can be produced as described inFIG. 7), by conversion of 5-hydroxypentanoate to 5-hydroxypentanal by apolypeptide having carboxylate reductase activity classified, forexample, under EC 1.2.99.6 such as from a Mycobacterium marinum (seeGenbank Accession No. ACC40567.1, SEQ ID NO: 2), a Mycobacteriumsmegmatis (see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), aSegniliparus rugosus (see Genbank Accession No. EFV11917.1, SEQ ID NO:4), a Mycobacterium massiliense (see Genbank Accession No. EIV11143.1,SEQ ID NO: 6), or a Segniliparus rotundus (see Genbank Accession No.ADG98140.1, SEQ ID NO: 7), in combination with a phosphopantetheinetransferase enhancer (e.g., encoded by a sfp (Genbank Accession No.CAA44858.1, SEQ ID NO:21) gene from Bacillus subtilis or npt (GenbankAccession No. ABI83656.1, SEQ ID NO:22) gene from Nocardia), or the geneproduct of GriC & GriD (Suzuki et al., J. Antibiot., 2007, 60(6),380-387); followed by conversion of 5-oxopentanol to 5-aminopentanol bya polypeptide having ω-transaminase activity classified, for example,under EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC2.6.1.48, or EC 2.6.1.82 such as from a Chromobacterium violaceum (seeGenbank Accession No. AAQ59697.1, SEQ ID NO: 8), a Pseudomonas syringae(see Genbank Accession No. AAY39893.1, SEQ ID NO: 10), a Rhodobactersphaeroides (see Genbank Accession No. ABA81135.1, SEQ ID NO: 11);followed by conversion to 5-aminopentanal by a polypeptide havingalcohol dehydrogenase activity classified, for example, under EC 1.1.1.-(e.g., EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as thegene product of YMR318C (Genbank Accession No. CAA90836.1) or YqhD (fromE. coli, GenBank Accession No. AAA69178.1) (Liu et al., Microbiology,2009, 155, 2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1),163-172; Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) orthe protein having GenBank Accession No. CAA81612.1; followed byconversion to cadaverine classified, for example, under EC 2.6.1.- suchas 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 suchas from a Chromobacterium violaceum (see Genbank Accession No.AAQ59697.1, SEQ ID NO: 8), a Pseudomonas aeruginosa (see GenbankAccession No. AAG08191.1, SEQ ID NO: 9), a Pseudomonas syringae (seeGenbank Accession No. AAY39893.1, SEQ ID NO: 10), or an Escherichia coli(see Genbank Accession No. AAA57874.1, SEQ ID NO: 12). See FIG. 6.

In some embodiments, cadaverine is synthesized from the centralprecursor 5-aminopentanoate by conversion of 5-aminopentanoate toN5-acetyl-5-aminopentanoate by a polypeptide having N-acetyltransferaseactivity such as a polypeptide having lysine N-acetyltransferaseactivity classified, for example, under EC 2.3.1.32; followed byconversion to N5-acetyl-5-aminopentanal by a polypeptide havingcarboxylate reductase activity such as from a Mycobacterium smegmatis(see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), a Mycobacteriummassiliense (see Genbank Accession No. EIV11143.1, SEQ ID NO: 6), or aSegniliparus rotundus (see Genbank Accession No. ADG98140.1, SEQ ID NO:7), in combination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp (Genbank Accession No. CAA44858.1, SEQ ID NO:21) genefrom Bacillus subtilis or npt (Genbank Accession No. ABI83656.1, SEQ IDNO:22) gene from Nocardia), or the gene product of GriC & GriD (Suzukiet al., J. Antibiot., 2007, 60(6), 380-387); followed by conversion toN5-acetyl-1,5-diaminopentane by a polypeptide having ω-transaminaseactivity such as the gene product of car in combination with aphosphopantetheine transferase enhancer (e.g., encoded by a sfp genefrom Bacillus subtilis or npt gene from Nocardia) or the gene productsof GriC and GriD from Streptomyces griseus; followed by conversion tocadaverine by an acetylputrescine deacetylase classified, for example,under EC 3.5.1.17 or EC 3.5.1.62. See, FIG. 6.

In some embodiments, cadaverine is synthesized from the centralprecursor glutarate semialdehyde by conversion of glutarate semialdehydeto pentanedial by a polypeptide having carboxylate reductase activityclassified, for example, under EC 1.2.99.6 such as from a Segniliparusrotundus (see Genbank Accession No. ADG98140.1, SEQ ID NO: 7), incombination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp (Genbank Accession No. CAA44858.1, SEQ ID NO:21) genefrom Bacillus subtilis or npt (Genbank Accession No. ABI83656.1, SEQ IDNO:22) gene from Nocardia), or the gene product of GriC & GriD (Suzukiet al., J. Antibiot., 2007, 60(6), 380-387); followed by conversion to5-aminopentanal by a polypeptide having ω-transaminase activityclassified, for example, under EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19,EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as from a Chromobacteriumviolaceum (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 8), aPseudomonas aeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO:9), a Pseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ IDNO: 10), a Rhodobacter sphaeroides (see Genbank Accession No.ABA81135.1, SEQ ID NO: 11), an Escherichia coli (see Genbank AccessionNo. AAA57874.1, SEQ ID NO: 12), or a Vibrio fluvialis (see GenbankAccession No. AEA39183.1, SEQ ID NO: 13); followed by conversion tocadaverine by a polypeptide having ω-transaminase activity classified,for example, under EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19, EC2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as from a Chromobacteriumviolaceum (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 8), aPseudomonas aeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO:9), a Pseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ IDNO: 10), or an Escherichia coli (see Genbank Accession No. AAA57874.1,SEQ ID NO: 12). See FIG. 6.

In some embodiments, cadaverine is synthesized from the centralprecursor 1,5-pentanediol by conversion of 1,5-pentanediol to5-hydroxypentanal by a polypeptide having alcohol dehydrogenase activityclassified, for example, under EC 1.1.1.- (e.g., EC 1.1.1.1, EC 1.1.1.2,EC 1.1.1.21, or EC 1.1.1.184) such as the gene product of YMR318C(Genbank Accession No. CAA90836.1) or YqhD (from E. coli, GenBankAccession No. AAA69178.1) (Liu et al., Microbiology, 2009, 155,2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; Jarboe,2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the proteinhaving GenBank Accession No. CAA81612.1; followed by conversion of5-oxopentanal to 5-aminopentanol by a polypeptide having ω-transaminaseactivity classified, for example, under EC 2.6.1.- such as 2.6.1.18, EC2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as from aChromobacterium violaceum (see Genbank Accession No. AAQ59697.1, SEQ IDNO: 8), a Pseudomonas aeruginosa (see Genbank Accession No. AAG08191.1,SEQ ID NO: 9), a Pseudomonas syringae (see Genbank Accession No.AAY39893.1, SEQ ID NO: 10), a Rhodobacter sphaeroides (see GenbankAccession No. ABA81135.1, SEQ ID NO: 11), an Escherichia coli (seeGenbank Accession No. AAA57874.1, SEQ ID NO: 12), or a Vibrio fluvialis(see Genbank Accession No. AEA39183.1, SEQ ID NO: 13); followed byconversion to 5-aminopentanal by a polypeptide having alcoholdehydrogenase activity classified, for example, under EC 1.1.1.- (e.g.,EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the geneproduct of YMR318C (Genbank Accession No. CAA90836.1) or YqhD (from E.coli, GenBank Accession No. AAA69178.1) (Liu et al., Microbiology, 2009,155, 2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172;Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or theprotein having GenBank Accession No. CAA81612.1; followed by conversionto cadaverine by a polypeptide having ω-transaminase activityclassified, for example, under EC 2.6.1.- such as 2.6.1.18, EC 2.6.1.19,EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as from a Chromobacteriumviolaceum (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 8), aPseudomonas aeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO:9), a Pseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ IDNO: 10), or an Escherichia coli (see Genbank Accession No. AAA57874.1,SEQ ID NO: 12). See FIG. 6.

Pathways Using 5-Hydroxypentanoate or Pentanoyl-CoA as Central Precursorto 1,5-Pentanediol

In some embodiments, 1,5 pentanediol is synthesized from the centralprecursor 5-hydroxypentanoate by conversion of 5-hydroxypentanoate to5-hydroxypentanal by a polypeptide having carboxylate reductase activityclassified, for example, under EC 1.2.99.6 such as from a Mycobacteriummarinum (see Genbank Accession No. ACC40567.1, SEQ ID NO: 2), aMycobacterium smegmatis (see Genbank Accession No. ABK71854.1, SEQ IDNO: 3), a Mycobacterium smegmatis (see Genbank Accession No. ABK75684.1,SEQ ID NO: 5), a Mycobacterium massiliense (see Genbank Accession No.EIV11143.1, SEQ ID NO: 6), or a Segniliparus rotundus (see GenbankAccession No. ADG98140.1, SEQ ID NO: 7), in combination with aphosphopantetheine transferase enhancer (e.g., encoded by a sfp (GenbankAccession No. CAA44858.1, SEQ ID NO:21) gene from Bacillus subtilis ornpt (Genbank Accession No. ABI83656.1, SEQ ID NO:22) gene fromNocardia), or the gene product of GriC & GriD (Suzuki et al., J.Antibiot., 2007, 60(6), 380-387); followed by conversion of5-hydroxypentanal to 1,7 pentanediol by a polypeptide having alcoholdehydrogenase activity classified, for example, under EC 1.1.1.- such asEC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the geneproduct of YMR318C (Genbank Accession No. CAA90836.1) or YqhD (from E.coli, GenBank Accession No. AAA69178.1) (see, e.g., Liu et al.,Microbiology, 2009, 155, 2078-2085; Larroy et al., 2002, Biochem J.,361(Pt 1), 163-172; or Jarboe, 2011, Appl. Microbiol. Biotechnol.,89(2), 249-257) or the protein having GenBank Accession No. CAA81612.1(from Geobacillus stearothermophilus). See, FIG. 8.

In some embodiments, pentanoyl-CoA can be converted to pentanoic acidpentyl ester using a polypeptide having alcohol O-acetyltransferaseactivity classified, for example, under EC 2.3.1.-(84) such as the geneproduct of Eht1 (Genbank Accession No: CAA85138.1, SEQ ID NO: 26);followed by conversion to pentanoic acid 5-hydroxy pentyl ester and/or5-hydroxypentanoic acid 5-hydroxypentyl ester using a polypeptide havingmonooxygenase activity classified, for example, under EC 1.14.14.- or EC1.14.15.- (1,3). For example, a polypeptide having monooxygenaseactivity can be, for example, from the CYP153A family, the CYP52A3(Genbank Accession No: BAA00371.1, SEQ ID NO: 29) family or the alkBfamily such as the gene product of alkBGT from Pseudomonas putida(Genbank Accession No. CAB51047.1, SEQ ID NO: 28); followed byconversion of pentanoic acid 5-hydroxy pentyl ester and/or5-hydroxypentanoic acid 5-hydroxypentyl to 1,5-pentanediol using apolypeptide having esterase activity classified, for example, under EC3.1.1.-(1,6) such as the gene product of EstC (see GenBank Accession No.AAC60471.2, SEQ ID NO: 27). See FIG. 8.

Cultivation Strategy

In some embodiments, the cultivation strategy entails achieving anaerobic, anaerobic, micro-aerobic, or mixed oxygen/denitrificationcultivation condition. Enzymes characterized in vitro as being oxygensensitive require a micro-aerobic cultivation strategy maintaining avery low dissolved oxygen concentration (See, for example, Chayabatra &Lu-Kwang, Appl. Environ. Microbiol., 2000, 66(2), 493 0 498; Wilson andBouwer, 1997, Journal of Industrial Microbiology and Biotechnology,18(2-3), 116-130).

In some embodiments, the cultivation strategy entails nutrientlimitation such as nitrogen, phosphate or oxygen limitation.

In some embodiments, a final electron acceptor other than oxygen such asnitrates can be utilized.

In some embodiments, a cell retention strategy using, for example,ceramic membranes can be employed to achieve and maintain a high celldensity during either fed-batch or continuous fermentation.

In some embodiments, the principal carbon source fed to the fermentationin the synthesis of one or more C5 building blocks can derive frombiological or non-biological feedstocks.

In some embodiments, the biological feedstock can be or can derive from,monosaccharides, disaccharides, lignocellulose, hemicellulose,cellulose, lignin, levulinic acid and formic acid, triglycerides,glycerol, fatty acids, agricultural waste, condensed distillers'solubles, or municipal waste.

The efficient catabolism of crude glycerol stemming from the productionof biodiesel has been demonstrated in several microorganisms such asEscherichia coli, Cupriavidus necator, Pseudomonas oleavorans,Pseudomonas putida and Yarrowia lipolytica (Lee et al., Appl. Biochem.Biotechnol., 2012, 166:1801-1813; Yang et al., Biotechnology forBiofuels, 2012, 5:13; Meijnen et al., Appl. Microbiol. Biotechnol.,2011, 90:885-893).

The efficient catabolism of lignocellulosic-derived levulinic acid hasbeen demonstrated in several organisms such as Cupriavidus necator andPseudomonas putida in the synthesis of 3-hydroxyvalerate via theprecursor propanoyl-CoA (Jaremko and Yu, 2011, supra; Martin andPrather, J. Biotechnol., 2009, 139:61-67).

The efficient catabolism of lignin-derived aromatic compounds such asbenzoate analogues has been demonstrated in several microorganisms suchas Pseudomonas putida, Cupriavidus necator (Bugg et al., Current Opinionin Biotechnology, 2011, 22, 394-400; Pérez-Pantoja et al., FEMSMicrobiol. Rev., 2008, 32, 736-794).

The efficient utilization of agricultural waste, such as olive millwaste water has been demonstrated in several microorganisms, includingYarrowia lipolytica (Papanikolaou et al., Bioresour. Technol., 2008,99(7):2419-2428).

The efficient utilization of fermentable sugars such as monosaccharidesand disaccharides derived from cellulosic, hemicellulosic, cane and beetmolasses, cassava, corn and other agricultural sources has beendemonstrated for several microorganism such as Escherichia coli,Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcuslactis (see, e.g., Hermann et al, J. Biotechnol., 2003, 104:155-172; Weeet al., Food Technol. Biotechnol., 2006, 44(2):163-172; Ohashi et al.,J. Bioscience and Bioengineering, 1999, 87(5):647-654).

The efficient utilization of furfural, derived from a variety ofagricultural lignocellulosic sources, has been demonstrated forCupriavidus necator (Li et al., Biodegradation, 2011, 22:1215-1225).

In some embodiments, the non-biological feedstock can be or can derivefrom natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate,non-volatile residue (NVR) or a caustic wash waste stream fromcyclohexane oxidation processes, or terephthalic acid/isophthalic acidmixture waste streams.

The efficient catabolism of methanol has been demonstrated for themethylotrophic yeast Pichia pastoris.

The efficient catabolism of ethanol has been demonstrated forClostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008,105(6) 2128-2133).

The efficient catabolism of CO₂ and H₂, which may be derived fromnatural gas and other chemical and petrochemical sources, has beendemonstrated for Cupriavidus necator (Prybylski et al., Energy,Sustainability and Society, 2012, 2:11).

The efficient catabolism of syngas has been demonstrated for numerousmicroorganisms, such as Clostridium ljungdahlii and Clostridiumautoethanogenum (Köpke et al., Applied and Environmental Microbiology,2011, 77(15):5467-5475).

The efficient catabolism of the non-volatile residue waste stream fromcyclohexane processes has been demonstrated for numerous microorganisms,such as Delftia acidovorans and Cupriavidus necator (Ramsay et al.,Applied and Environmental Microbiology, 1986, 52(1):152-156).

In some embodiments, the host microorganism is a prokaryote. Forexample, the prokaryote can be a bacterium from the genus Escherichiasuch as Escherichia coli; from the genus Clostridia such as Clostridiumljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; fromthe genus Corynebacteria such as Corynebacterium glutamicum; from thegenus Cupriavidus such as Cupriavidus necator or Cupriavidusmetallidurans; from the genus Pseudomonas such as Pseudomonasfluorescens, Pseudomonas putida or Pseudomonas oleavorans; from thegenus Delftia such as Delftia acidovorans; from the genus Bacillus suchas Bacillus subtillis; from the genus Lactobacillus such asLactobacillus delbrueckii; or from the genus Lactococcus such asLactococcus lactis. Such prokaryotes also can be a source of genes toconstruct recombinant host cells described herein that are capable ofproducing one or more C5 building blocks.

In some embodiments, the host microorganism is a eukaryote. For example,the eukaryote can be a filamentous fungus, e.g., one from the genusAspergillus such as Aspergillus niger. Alternatively, the eukaryote canbe a yeast, e.g., one from the genus Saccharomyces such as Saccharomycescerevisiae; from the genus Pichia such as Pichia pastoris; or from thegenus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkiasuch as Issathenkia orientalis; from the genus Debaryomyces such asDebaryomyces hansenii; from the genus Arxula such as Arxulaadenoinivorans; or from the genus Kluyveromyces such as Kluyveromyceslactis. Such eukaryotes also can be a source of genes to constructrecombinant host cells described herein that are capable of producingone or more C5 building blocks.

Metabolic Engineering

The present document provides methods involving less than all the stepsdescribed for all the above pathways. Such methods can involve, forexample, one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve or more of such steps. Where less than all the steps areincluded in such a method, the first, and in some embodiments the only,step can be any one of the steps listed.

Furthermore, recombinant hosts described herein can include anycombination of the above enzymes such that one or more of the steps,e.g., one, two, three, four, five, six, seven, eight, nine, ten, or moreof such steps, can be performed within a recombinant host. This documentprovides host cells of any of the genera and species listed andgenetically engineered to express one or more (e.g., two, three, four,five, six, seven, eight, nine, 10, 11, 12 or more) recombinant forms ofany of the enzymes recited in the document. Thus, for example, the hostcells can contain exogenous nucleic acids encoding enzymes catalyzingone or more of the steps of any of the pathways described herein.

In addition, this document recognizes that where enzymes have beendescribed as accepting CoA-activated substrates, analogous enzymeactivities associated with [acp]-bound substrates exist that are notnecessarily in the same enzyme class.

Also, this document recognizes that where enzymes have been describedaccepting (R)-enantiomers of substrate, analogous enzyme activitiesassociated with (S)-enantiomer substrates exist that are not necessarilyin the same enzyme class.

This document also recognizes that where an enzyme is shown to accept aparticular co-factor, such as NADPH, or co-substrate, such asacetyl-CoA, many enzymes are promiscuous in terms of accepting a numberof different co-factors or co-substrates in catalyzing a particularenzyme activity. Also, this document recognizes that where enzymes havehigh specificity for e.g., a particular co-factor such as NADH, anenzyme with similar or identical activity that has high specificity forthe co-factor NADPH may be in a different enzyme class. In addition,enzymes in a pathway that require a particular co-factor can be replacedwith an enzyme that has similar or identical activity and specificityfor a different co-factor. For example, one or more steps in a pathwaythat use an enzyme with specificity for NADH can be replaced with anenzyme having similar or identical activity and specificity for NADPH.Similarly, one or more steps in a pathway that use an enzyme withspecificity for NADPH can be replaced with an enzyme having similar oridentical activity and specificity for NADH.

In some embodiments, the enzymes in the pathways outlined herein are theresult of enzyme engineering via non-direct or rational enzyme designapproaches with aims of improving activity, improving specificity,reducing feedback inhibition, reducing repression, improving enzymesolubility, changing stereo-specificity, or changing co-factorspecificity.

In some embodiments, the enzymes in the pathways outlined here can begene dosed, i.e., overexpressed, into the resulting genetically modifiedorganism via episomal or chromosomal integration approaches.

In some embodiments, genome-scale system biology techniques such as FluxBalance Analysis can be utilized to devise genome scale attenuation orknockout strategies for directing carbon flux to a C5 building block.

Attenuation strategies include, but are not limited to; the use oftransposons, homologous recombination (double cross-over approach),mutagenesis, enzyme inhibitors and RNAi interference.

In some embodiments, fluxomic, metabolomic and transcriptomal data canbe utilized to inform or support genome-scale system biology techniques,thereby devising genome scale attenuation or knockout strategies indirecting carbon flux to a C5 building block.

In some embodiments, the host microorganism's tolerance to highconcentrations of a C5 building block can be improved through continuouscultivation in a selective environment.

In some embodiments, the host microorganism's endogenous biochemicalnetwork can be attenuated or augmented to (1) ensure the intracellularavailability of acetyl-CoA and propanoyl-CoA, (2) create a NADH or NADPHimbalance that may be balanced via the formation of one or more C5building blocks, (3) prevent degradation of central metabolites, centralprecursors leading to and including one or more C5 building blocksand/or (4) ensure efficient efflux from the cell.

In some embodiments requiring intracellular availability ofpropanoyl-CoA for C5 building block synthesis, endogenous enzymescatalyzing the hydrolysis of propionoyl-CoA and acetyl-CoA such asshort-chain length polypeptides having thioesterase activity can beattenuated in the host organism.

In some embodiments requiring the intracellular availability ofpropanoyl-CoA for C5 building block synthesis, endogenous enzymesconsuming propanoyl-CoA to succinyl-CoA via the methylcitrate cycle suchas a polypeptide having methylcitrate synthase activity can beattenuated in the host organism (Bramer & Steinbüchel, 2001,Microbiology, 147: 2203-2214).

In some embodiments requiring the intracellular availability ofpropanoyl-CoA via L-threonine as central metabolite for C5 buildingblock synthesis, a feedback-resistant polypeptide having threoninedeaminase activity can be genetically engineered into the host organism(Tseng et al., Microbial Cell Factories, 2010, 9:96).

In some embodiments requiring condensation of acetyl-CoA andpropanoyl-CoA for C5 building block synthesis, one or more endogenouspolypeptides having β-ketothiolases activity catalyzing the condensationof only acetyl-CoA to acetoacetyl-CoA such as the endogenous geneproducts of AtoB or phaA can be attenuated.

In some embodiments requiring the intracellular availability ofacetyl-CoA for C5 building block synthesis, an endogenous polypeptidehaving phosphotransacetylase activity generating acetate such as pta canbe attenuated (Shen et al., Appl. Environ. Microbiol., 2011,77(9):2905-2915).

In some embodiments requiring the intracellular availability ofacetyl-CoA for C5 building block synthesis, an endogenous gene in anacetate synthesis pathway encoding a polypeptide having acetate kinaseactivity, such as ack, can be attenuated.

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C5 building block synthesis, an endogenous geneencoding an enzyme that catalyzes the degradation of pyruvate to lactatesuch as a polypeptide having lactate dehydrogenase activity encoded byIdhA can be attenuated (Shen et al., 2011, supra).

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C5 building block synthesis, endogenous genesencoding enzymes, such as a polypeptide having menaquinol-fumarateoxidoreductase activity, that catalyze the degradation ofphophoenolpyruvate to succinate such as frdBC can be attenuated (see,e.g., Shen et al., 2011, supra).

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C5 building block synthesis, an endogenous geneencoding an enzyme that catalyzes the degradation of acetyl-CoA toethanol such as the polypeptide having alcohol dehydrogenase activityencoded by adhE can be attenuated (Shen et al., 2011, supra).

In some embodiments, where pathways require excess NADH co-factor for C5building block synthesis, a recombinant polypeptide having formatedehydrogenase activity can be overexpressed in the host organism (Shenet al., 2011, supra).

In some embodiments, where pathways require excess NADH or NADPHco-factor for C5 building block synthesis, a polypeptide havingtranshydrogenase activity dissipating the cofactor imbalance can beattenuated.

In some embodiments, an endogenous gene encoding an enzyme thatcatalyzes the degradation of pyruvate to ethanol such as polypeptidehaving pyruvate decarboxylase activity can be attenuated.

In some embodiments, an endogenous gene encoding an enzyme thatcatalyzes the generation of isobutanol such as a polypeptide having2-oxoacid decarboxylase activity can be attenuated.

In some embodiments requiring the intracellular availability ofacetyl-CoA for C5 building block synthesis, a recombinant polypeptidehaving acetyl-CoA synthetase activity such as the gene product of acscan be overexpressed in the microorganism (Satoh et al., J. Bioscienceand Bioengineering, 2003, 95(4):335-341).

In some embodiments, carbon flux can be directed into the pentosephosphate cycle to increase the supply of NADPH by attenuating anendogenous polypeptide having glucose-6-phosphate isomerase activity (EC5.3.1.9).

In some embodiments, carbon flux can be redirected into the pentosephosphate cycle to increase the supply of NADPH by overexpression apolypeptide having 6-phosphogluconate dehydrogenase activity and/or apolypeptide having transketolase activity (Lee et al., 2003,Biotechnology Progress, 19(5), 1444-1449).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C5 building block, a gene such as UdhA encoding apolypeptide having puridine nucleotide transhydrogenase activity can beoverexpressed in the host organisms (Brigham et al., Advanced Biofuelsand Bioproducts, 2012, Chapter 39, 1065-1090).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C5 Building Block, a recombinant polypeptide havingglyceraldehyde-3-phosphate-dehydrogenase activity such as GapN can beoverexpressed in the host organisms (Brigham et al., 2012, supra).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C5 building block, a recombinant polypeptide havingmalic enzyme activity such as maeA or maeB can be overexpressed in thehost organisms (Brigham et al., 2012, supra).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C5 building block, a recombinant polypeptide havingglucose-6-phosphate dehydrogenase activity such as zwf can beoverexpressed in the host organisms (Lim et al., J. Bioscience andBioengineering, 2002, 93(6), 543-549).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C5 building block, a recombinant polypeptide havingfructose 1,6 diphosphatase activity such as fbp can be overexpressed inthe host organisms (Becker et al., J. Biotechnol., 2007, 132:99-109).

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C5 building block, endogenous polypeptide havingtriose phosphate isomerase activity (EC 5.3.1.1) can be attenuated.

In some embodiments, where pathways require excess NADPH co-factor inthe synthesis of a C5 building block, a recombinant polypeptide havingglucose dehydrogenase activity such as the gene product of gdh can beoverexpressed in the host organism (Satoh et al., J. Bioscience andBioengineering, 2003, 95(4):335-341).

In some embodiments, endogenous enzymes facilitating the conversion ofNADPH to NADH can be attenuated, such as the NADH generation cycle thatmay be generated via inter-conversion of polypeptides having glutamatedehydrogenase activity classified under EC 1.4.1.2 (NADH-specific) andEC 1.4.1.4 (NADPH-specific).

In some embodiments, an endogenous polypeptide having glutamatedehydrogenase activity (EC 1.4.1.3) that utilizes both NADH and NADPH asco-factors can be attenuated.

In some embodiments, a membrane-bound polypeptide having cytochrome P450activity such as CYP4F3B can be solubilized by only expressing thecytosolic domain and not the N-terminal region that anchors the P450 tothe endoplasmic reticulum (see, for example, Scheller et al., J. Biol.Chem., 1994, 269(17):12779-12783).

In some embodiments, a membrane-bound polypeptide having enoyl-CoAreductase activity can be solubilized via expression as a fusion proteinto a small soluble protein such as a maltose binding protein (Gloerichet al., FEBS Letters, 2006, 580, 2092-2096).

In some embodiments using hosts that naturally accumulatepolyhydroxyalkanoates, the endogenous polypeptide havingpolyhydroxyalkanoate synthase activity can be attenuated in the hoststrain.

In some embodiments requiring the intracellular availability ofpentanoyl-CoA for C5 building block synthesis, a recombinant polypeptidehaving propionyl-CoA synthetase activity such as the gene product ofPrpE-RS can be overexpressed in the microorganism (Rajashekhara &Watanabe, FEBS Letters, 2004, 556:143-147).

In some embodiments, a polypeptide having L-alanine dehydrogenaseactivity can be overexpressed in the host to regenerate L-alanine frompyruvate as an amino donor for ω-transaminase reactions.

In some embodiments, a polypeptide having L-glutamate dehydrogenaseactivity, a polypeptide having L-glutamine synthetase activity, or apolypeptide having glutamate synthase activity can be overexpressed inthe host to regenerate L-glutamate from 2-oxoglutarate as an amino donorfor ω-transaminase reactions.

In some embodiments, enzymes such as polypeptide having pimeloyl-CoAdehydrogenase activity classified under, EC 1.3.1.62; a polypeptidehaving acyl-CoA dehydrogenase activity classified, for example, under EC1.3.8.7 or EC 1.3.8.1; and/or a polypeptide having glutaryl-CoAdehydrogenase activity classified, for example, under EC 1.3.8.6 thatdegrade central metabolites and central precursors leading to andincluding C5 building blocks can be attenuated.

In some embodiments, endogenous enzymes activating C5 building blocksvia Coenzyme A esterification such as polypeptides having CoA-ligaseactivity (e.g., a pimeloyl-CoA synthetase) classified under, forexample, EC 6.2.1.14 can be attenuated.

In some embodiments, the efflux of a C5 building block across the cellmembrane to the extracellular media can be enhanced or amplified bygenetically engineering structural modifications to the cell membrane orincreasing any associated transporter activity for a C5 building block.

The efflux of cadaverine can be enhanced or amplified by overexpressingbroad substrate range multidrug transporters such as Blt from Bacillussubtilis (Woolridge et al., 1997, J. Biol. Chem., 272(14):8864-8866);AcrB and AcrD from Escherichia coli (Elkins & Nikaido, 2002, J.Bacteriol., 184(23), 6490-6499), NorA from Staphylococcus aereus (Ng etal., 1994, Antimicrob Agents Chemother, 38(6), 1345-1355), or Bmr fromBacillus subtilis (Neyfakh, 1992, Antimicrob Agents Chemother, 36(2),484-485).

The efflux of 5-aminopentanoate and cadaverine can be enhanced oramplified by overexpressing the solute transporters such as the lysEtransporter from Corynebacterium glutamicum (Bellmann et al., 2001,Microbiology, 147, 1765-1774).

The efflux of glutaric acid can be enhanced or amplified byoverexpressing a dicarboxylate transporter such as the SucE transporterfrom Corynebacterium glutamicum (Huhn et al., Appl. Microbiol. &Biotech., 89(2), 327-335).

Producing C5 Building Blocks Using a Recombinant Host

Typically, one or more C5 building blocks can be produced by providing ahost microorganism and culturing the provided microorganism with aculture medium containing a suitable carbon source as described above.In general, the culture media and/or culture conditions can be such thatthe microorganisms grow to an adequate density and produce a C5 buildingblock efficiently. For large-scale production processes, any method canbe used such as those described elsewhere (Manual of IndustrialMicrobiology and Biotechnology, 2^(nd) Edition, Editors: A. L. Demainand J. E. Davies, ASM Press; and Principles of Fermentation Technology,P. F. Stanbury and A. Whitaker, Pergamon). Briefly, a large tank (e.g.,a 100 gallon, 200 gallon, 500 gallon, or more tank) containing anappropriate culture medium is inoculated with a particularmicroorganism. After inoculation, the microorganism is incubated toallow biomass to be produced. Once a desired biomass is reached, thebroth containing the microorganisms can be transferred to a second tank.This second tank can be any size. For example, the second tank can belarger, smaller, or the same size as the first tank. Typically, thesecond tank is larger than the first such that additional culture mediumcan be added to the broth from the first tank. In addition, the culturemedium within this second tank can be the same as, or different from,that used in the first tank.

Once transferred, the microorganisms can be incubated to allow for theproduction of a C5 building block. Once produced, any method can be usedto isolate C5 building blocks. For example, C5 building blocks can berecovered selectively from the fermentation broth via adsorptionprocesses. In the case of glutaric acid and 5-aminopentanoic acid, theresulting eluate can be further concentrated via evaporation,crystallized via evaporative and/or cooling crystallization, and thecrystals recovered via centrifugation. In the case of cadaverine and1,5-pentanediol, distillation may be employed to achieve the desiredproduct purity.

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

EXAMPLES Example 1

Enzyme Activity of ω-Transaminase Using Glutarate Semialdehyde asSubstrate and Forming 5-Aminopentanoate

A nucleotide sequence encoding an N-terminal His-tag was added to thegenes from Chromobacterium violaceum and Rhodobacter sphaeroidesencoding the ω-transaminases of SEQ ID NOs: 8 and 10 respectively (seeFIG. 10) such that N-terminal HIS tagged ω-transaminases could beproduced. Each of the resulting modified genes was cloned into a pET21aexpression vector under control of the T7 promoter and each expressionvector was transformed into a BL21[DE3] E. coli host. The resultingrecombinant E. coli strains were cultivated at 37° C. in a 250 mL shakeflask culture containing 50 mL LB media and antibiotic selectionpressure, with shaking at 230 rpm. Each culture was induced overnight at16 OC using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., 5-aminopentanoateto glutarate semialdehyde) were performed in a buffer composed of afinal concentration of 50 mM HEPES buffer (pH=7.5), 10 mM5-aminopentanoate, 10 mM pyruvate and 100 μM pyridoxyl 5′ phosphate.Each enzyme activity assay reaction was initiated by adding cell freeextract of the ω-transaminase gene product or the empty vector controlto the assay buffer containing the 5-aminopentanoate and incubated at25° C. for 4 h, with shaking at 250 rpm. The formation of L-alanine frompyruvate was quantified via RP-HPLC.

Each enzyme only control without 5-aminopentanoate demonstrated low baseline conversion of pyruvate to L-alanine. See FIG. 17. The gene productof SEQ ID NO 8, accepted 5-aminopentanoate as substrate as confirmedagainst the empty vector control. See FIG. 18.

Enzyme activity in the forward direction (i.e., glutarate semialdehydeto 5-aminopentanoate) was confirmed for the transaminase of SEQ ID NO10. Enzyme activity assays were performed in a buffer composed of afinal concentration of 50 mM HEPES buffer (pH=7.5), 10 mM glutaratesemialdehyde, 10 mM L-alanine and 100 μM pyridoxyl 5′ phosphate. Eachenzyme activity assay reaction was initiated by adding a cell freeextract of the ω-transaminase gene product or the empty vector controlto the assay buffer containing the glutarate semialdehyde and incubatedat 25° C. for 4 h, with shaking at 250 rpm. The formation of pyruvatewas quantified via RP-HPLC.

The gene product of SEQ ID NO 10 accepted glutarate semialdehyde assubstrate as confirmed against the empty vector control. See FIG. 19.The reversibility of the ω-transaminase activity was confirmed,demonstrating that the ω-transaminases of SEQ ID NO 8, and SEQ ID NO 10accepted glutarate semialdehyde as substrate and synthesized5-aminopentanoate as a reaction product.

Example 2

Enzyme Activity of Carboxylate Reductase Using 5-Hydroxypentanoate asSubstrate and Forming 5-Hydroxypentanal

A nucleotide sequence encoding a His-tag was added to the genes fromMycobacterium marinum, Mycobacterium smegmatis, Segniliparus rugosus,Mycobacterium massiliense, and Segniliparus rotundus that encode thecarboxylate reductases of SEQ ID NOs: 2-4, 6 and 7, respectively(GenBank Accession Nos. ACC40567.1, ABK71854.1, EFV11917.1, EIV11143.1,and ADG98140.1, respectively) (see FIG. 10) such that N-terminal HIStagged carboxylate reductases could be produced. Each of the modifiedgenes was cloned into a pET Duet expression vector alongside a sfp geneencoding a His-tagged phosphopantetheine transferase from Bacillussubtilis, both under control of the T7 promoter. Each expression vectorwas transformed into a BL21[DE3] E. coli host along with the expressionvectors from Example 3. Each resulting recombinant E. coli strain wascultivated at 37° C. in a 250 mL shake flask culture containing 50 mL LBmedia and antibiotic selection pressure, with shaking at 230 rpm. Eachculture was induced overnight at 37° C. using an auto-induction media.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugation.The carboxylate reductases and phosphopantetheine transferase werepurified from the supernatant using Ni-affinity chromatography, diluted10-fold into 50 mM HEPES buffer (pH=7.5) and concentrated viaultrafiltration.

Enzyme activity (i.e., 5-hydroxypentanoate to 5-hydroxypentanal) assayswere performed in triplicate in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 2 mM 5-hydroxypentanal, 10mM MgCl₂, 1 mM ATP, and 1 mM NADPH. Each enzyme activity assay reactionwas initiated by adding purified carboxylate reductase andphosphopantetheine transferase or the empty vector control to the assaybuffer containing the 5-hydroxypentanoate and then incubated at roomtemperature for 20 min. The consumption of NADPH was monitored byabsorbance at 340 nm. Each enzyme only control without5-hydroxypentanoate demonstrated low base line consumption of NADPH. SeeFIG. 12.

The gene products of SEQ ID NOs: 2-4, 6 and 7, enhanced by the geneproduct of sfp, accepted 5-hydroxypentanoate as substrate as confirmedagainst the empty vector control (see FIG. 14), and synthesized5-hydroxypentanal.

Example 3

Enzyme Activity of ω-Transaminase for 5-Aminopentanol, Forming5-Oxopentanol

A nucleotide sequence encoding an N-terminal His-tag was added to theChromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae,Rhodobacter sphaeroide, Escherichia coli and Vibrio fluvialis genesencoding the ω-transaminases of SEQ ID NOs: 8-13, respectively (see FIG.10) such that N-terminal HIS tagged ω-transaminases could be produced.The modified genes were cloned into a pET21a expression vector under theT7 promoter. Each expression vector was transformed into a BL21[DE3] E.coli host. Each resulting recombinant E. coli strain were cultivated at37° C. in a 250 mL shake flask culture containing 50 mL LB media andantibiotic selection pressure, with shaking at 230 rpm. Each culture wasinduced overnight at 16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., 5-aminopentanolto 5-oxopentanol) were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM 5-aminopentanol, 10mM pyruvate, and 100 μM pyridoxyl 5′ phosphate. Each enzyme activityassay reaction was initiated by adding cell free extract of theω-transaminase gene product or the empty vector control to the assaybuffer containing the 5-aminopentanol and then incubated at 25° C. for 4h, with shaking at 250 rpm. The formation of L-alanine was quantifiedvia RP-HPLC.

Each enzyme only control without 5-aminopentanol had low base lineconversion of pyruvate to L-alanine. See FIG. 17.

The gene products of SEQ ID NOs: 8-13 accepted 5-aminopentanol assubstrate as confirmed against the empty vector control (see FIG. 13)and synthesized 5-oxopentanol as reaction product. Given thereversibility of the ω-transaminase activity (see Example 1), it can beconcluded that the gene products of SEQ ID NOs: 8-13 accept5-oxopentanol as substrate and form 5-aminopentanol.

Example 4

Enzyme Activity of ω-Transaminase Using Cadaverine as Substrate andForming 5-Aminopentanal

A nucleotide sequence encoding an N-terminal His-tag was added to theChromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae,and Escherichia coli genes encoding the ω-transaminases of SEQ ID NOs:8-10 and 12, respectively (see FIG. 10) such that N-terminal HIS taggedω-transaminases could be produced. The modified genes were cloned into apET21a expression vector under the T7 promoter. Each expression vectorwas transformed into a BL21[DE3] E. coli host. Each resultingrecombinant E. coli strain were cultivated at 37° C. in a 250 mL shakeflask culture containing 50 mL LB media and antibiotic selectionpressure, with shaking at 230 rpm. Each culture was induced overnight at16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., cadaverine to5-aminopentanal) were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM cadaverine, 10 mMpyruvate, and 100 μM pyridoxyl 5′ phosphate. Each enzyme activity assayreaction was initiated by adding cell free extract of the ω-transaminasegene product or the empty vector control to the assay buffer containingthe cadaverine and then incubated at 25° C. for 4 h, with shaking at 250rpm. The formation of L-alanine was quantified via RP-HPLC.

Each enzyme only control without cadaverine had low base line conversionof pyruvate to L-alanine. See FIG. 17.

The gene products of SEQ ID NOs: 8-10 and 12 accepted cadaverine assubstrate as confirmed against the empty vector control (see FIG. 20)and synthesized 5-aminopentanal as reaction product. Given thereversibility of the ω-transaminase activity (see Example 1), it can beconcluded that the gene products of SEQ ID NOs: 8-10 and 12 accept5-aminopentanal as substrate and form cadaverine.

Example 5

Enzyme Activity of ω-Transaminase Using N5-Acetyl-1,5-Diaminopentane,and Forming N5-Acetyl-5-Aminopentanal

The activity of the N-terminal His-tagged ω-transaminases of SEQ ID NOs:8, 10-13 (see Example 3, and FIG. 10) for convertingN5-acetyl-1,5-diaminopentane to N5-acetyl-5-aminopentanal was assayedusing a buffer composed of a final concentration of 50 mM HEPES buffer(pH=7.5), 10 mM N5-acetyl-1,5-diaminopentane, 10 mM pyruvate and 100 μMpyridoxyl 5′ phosphate. Each enzyme activity assay reaction wasinitiated by adding a cell free extract of the ω-transaminase or theempty vector control to the assay buffer containing theN5-acetyl-1,5-diaminopentane then incubated at 25° C. for 4 h, withshaking at 250 rpm. The formation of L-alanine was quantified viaRP-HPLC.

Each enzyme only control without N5-acetyl-1,5-diaminopentanedemonstrated low base line conversion of pyruvate to L-alanine. See FIG.17.

The gene product of SEQ ID NOs: 8, 10 acceptedN5-acetyl-1,5-diaminopentane as substrate as confirmed against the emptyvector control (see FIG. 15) and synthesized N5-acetyl-5-aminopentanalas reaction product.

Given the reversibility of the ω-transaminase activity (see Example 1),the gene products of SEQ ID NOs: 8, 10 accept N5-acetyl-5-aminopentanalas substrate forming N5-acetyl-1,5-diaminopentane.

Example 6

Enzyme Activity of Carboxylate Reductase Using Glutarate Semialdehyde asSubstrate and Forming Pentanedial

The N-terminal His-tagged carboxylate reductase of SEQ ID NO 7 (seeExample 3 and FIG. 10) was assayed using glutarate semialdehyde assubstrate. The enzyme activity assay was performed in triplicate in abuffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5),2 mM glutarate semialdehyde, 10 mM MgCl₂, 1 mM ATP and 1 mM NADPH. Theenzyme activity assay reaction was initiated by adding purifiedcarboxylate reductase and phosphopantetheine transferase or the emptyvector control to the assay buffer containing the glutarate semialdehydeand then incubated at room temperature for 20 min. The consumption ofNADPH was monitored by absorbance at 340 nm. The enzyme only controlwithout glutarate semialdehyde demonstrated low base line consumption ofNADPH. See FIG. 12.

The gene product of SEQ ID NO 7, enhanced by the gene product of sfp,accepted glutarate semialdehyde as substrate as confirmed against theempty vector control (see FIG. 16) and synthesized pentanedial.

Example 7

Enzyme Activity of Carboxylate Reductase Using Pentanoate as Substratein Forming Pentanal

The N-terminal His-tagged carboxylate reductase of SEQ ID NOs 2, 3, 6and 7 (see Example 3 and FIG. 10) was assayed using glutaratesemialdehyde as substrate. The enzyme activity assay was performed intriplicate in a buffer composed of a final concentration of 50 mM HEPESbuffer (pH=7.5), 2 mM pentanoate, 10 mM MgCl₂, 1 mM ATP and 1 mM NADPH.The enzyme activity assay reaction was initiated by adding purifiedcarboxylate reductase and phosphopantetheine transferase or the emptyvector control to the assay buffer containing the pentanoate and thenincubated at room temperature for 20 min. The consumption of NADPH wasmonitored by absorbance at 340 nm. The enzyme only control withoutpentanoate demonstrated low base line consumption of NADPH. See FIG. 12.

The gene products of SEQ ID NOs 2, 3, 6 and 7, enhanced by the geneproduct of sfp, accepted pentanoate as substrate as confirmed againstthe empty vector control (see FIG. 11) and synthesized pentanal.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of producing a terminal hydroxyl (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester in a recombinant host, said methodcomprising: a) enzymatically converting a C₄₋₉ carboxylic acid to a(C₃₋₈ alkyl)-C(═O)OCH₃ ester using a polypeptide having fatty acidO-methyltransferase activity, wherein said polypeptide having fatty acidO-methyltransferase activity is classified under EC 2.1.1.15 and has atleast 85% sequence identity to an amino acid sequence set forth in SEQID NO:23, SEQ ID NO:24, or SEQ ID NO:25; and b) enzymatically convertingthe (C₃₋₈ alkyl)-C(═O)OCH₃ ester to a terminal hydroxyl (C₃₋₈hydroxyalkyl)-C(═O)OCH₃ ester using a polypeptide having monooxygenaseactivity, wherein said polypeptide having monooxygenase activity isclassified under EC 1.14.14.- or EC 1.14.15.- and has at least 85%sequence identity to an amino acid sequence set forth in SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:28, or SEQ ID NO:29, the methodoptionally further comprising enzymatically converting the terminalhydroxyl (C₃₋₈ hydroxyalkyl)-C(═O)OCH₃ ester to a terminal hydroxyl C₄₋₉hydroxyalkanoate.
 2. The method of claim 1, wherein the C₄₋₉ carboxylicacid is enzymatically produced from a C₄₋₉alkanoyl-CoA using: apolypeptide having thioesterase activity, wherein said polypeptidehaving thioesterase activity is classified under EC 3.1.2.- and has atleast 85% sequence identity to the amino acid sequence set forth in SEQID NO:1, SEQ ID NO: 33, or SEQ ID NO: 34; or a polypeptide havingbutanal dehydrogenase activity and a polypeptide having aldehydedehydrogenase activity, wherein said polypeptide having butanaldehydrogenase activity is classified under EC 1.2.1.10 or EC 1.2.1.57and has at least 85% sequence identity to the amino acid sequence setforth in SEQ ID NO: 30 and said polypeptide having aldehydedehydrogenase activity is classified under EC 1.2.1.3 or EC 1.2.1.4. 3.The method of claim 1, wherein a polypeptide having demethylase activityclassified under EC 2.1.1.- or a polypeptide having esterase activityclassified under EC 3.1.1.- enzymatically converts the terminal hydroxyl(C₃₋₈ hydroxyalkyl)-C(═O)OCH₃ ester to a terminal hydroxyl C₄₋₉hydroxyalkanoate.
 4. The method of claim 1, wherein the C₄₋₉ carboxylicacid is pentanoate, and is enzymatically converted to pentanoate methylester; and the pentanoate methyl ester is enzymatically converted to5-hydroxypentanoate methyl ester, the method optionally furthercomprising enzymatically converting 5-hydroxypentanoate methyl ester to5-hydroxypentanoate using a polypeptide having demethylase activityclassified under EC 2.1.1.- or a polypeptide having esterase activityclassified under EC 3.1.1.-.
 5. The method of claim 2, wherein the C₄₋₉alkanoyl-CoA is pentanoyl-CoA and pentanoate is enzymatically producedfrom pentanoyl-CoA.
 6. A method of producing one or more terminalhydroxy-substituted (C₄₋₉alkyl)-OC(═O)—(C₃₋₈alkyl) esters in arecombinant host, said method comprising: a) enzymatically converting aC₄₋₉ alkanoyl-CoA to a (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester in saidhost using a polypeptide having alcohol O-acetyltransferase activity,wherein said polypeptide having alcohol O-acetyltransferase activity isclassified under EC 2.3.1.84 and has at least 85% sequence identity tothe amino acid sequence set forth in SEQ ID NO: 26; and b) enzymaticallyconverting the (C₄₋₉alkyl)-OC(═O)—(C₃₋₈alkyl) ester to any of (C₄₋₉alkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester, (C₄₋₉hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester, or (C₄₋₉hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester in said host, the methodoptionally further comprising enzymatically converting (C₄₋₉hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester or (C₄₋₉alkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester to a terminal hydroxyl C₄₋₉hydroxyalkanoate, wherein the enzymatic conversion uses a polypeptidehaving esterase activity to convert (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈hydroxyalkyl) ester or (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈alkyl) ester tothe C₄₋₉ hydroxyalkanoate, wherein said polypeptide having esteraseactivity is classified under EC 3.1.1.- and has at least 85% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:
 27. 7. Themethod of claim 6, wherein (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈ alkyl) ester isenzymatically converted to any of (C₄₋₉ alkyl)-OC(═O)—(C₃₋₈hydroxyalkyl) ester, (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl)ester, or (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈alkyl) ester using apolypeptide having monooxygenase activity, wherein said polypeptidehaving monooxygenase activity is classified under EC 1.14.14.- or EC1.14.15.- and has at least 85% sequence identity to an amino acidsequence set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQID NO:28, or SEQ ID NO:29.
 8. The method of claim 6, wherein the C₄₋₉alkanoyl-CoA is pentanoyl-CoA and is enzymatically converted topentanoic acid pentyl ester; and pentanoic acid pentyl ester isenzymatically converted to any of 5-hydroxypentanoic acid pentyl ester,5-hydroxypentanoic acid 5-hydroxypentyl ester, or pentanoic acid5-hydroxypentyl ester using a polypeptide having monooxygenase activityclassified under EC 1.14.14.- or EC 1.14.15.-, the method optionallyfurther comprising enzymatically converting 5-hydroxypentanoic acid5-hydroxypentyl ester or 5-hydroxypentanoic acid pentyl ester to5-hydroxypentanoate, said enzymatic conversion of 5-hydroxypentanoicacid 5-hydroxypentyl ester or 5-hydroxypentanoic acid pentyl ester to5-hydroxypentanoate using a polypeptide having esterase activityclassified under EC 3.1.1.-.
 9. The method of claim 8, said methodfurther comprising enzymatically converting 5-hydroxypentanoic acid5-hydroxypentyl ester or pentanoic acid 5-hydroxypentyl ester to1,5-pentanediol, said enzymatic conversion of 5-hydroxypentanoic acid5-hydroxypentyl ester or pentanoic acid 5-hydroxypentyl ester to1,5-pentanediol using a polypeptide having esterase activity classifiedunder EC 3.1.1.-, the method optionally further comprising enzymaticallyconverting 1,5-pentanediol to 5-hydroxypentanoate using a polypeptidehaving alcohol dehydrogenase activity classified under EC 1.1.1- and apolypeptide having aldehyde dehydrogenase activity classified under EC1.2.1.3 or EC 1.2.1.4.
 10. The method of claim 8, wherein the methodfurther comprises enzymatically converting 5-hydroxypentanoic acid5-hydroxypentyl ester or 5-hydroxypentanoic acid pentyl ester to5-hydroxypentanoate, said enzymatic conversion of 5-hydroxypentanoicacid 5-hydroxypentyl ester or 5-hydroxypentanoic acid pentyl ester to5-hydroxypentanoate using a polypeptide having esterase activityclassified under EC 3.1.1.-, said method further comprisingenzymatically converting 5-hydroxypentanoate to glutarate semialdehyde,said enzymatic conversion of 5-hydroxypentanoate to glutaratesemialdehyde using: a polypeptide having alcohol dehydrogenase activityclassified under EC 1.1.1.-, a polypeptide having 6-hydroxyhexanoatedehydrogenase activity classified under EC 1.1.1.258, or a polypeptidehaving cytochrome P450 monooxygenase activity classified under EC1.14.14.-, the method optionally further comprising enzymaticallyconverting glutarate semialdehyde to glutaric acid using a polypeptidehaving 7-oxoheptanoate dehydrogenase activity classified under EC1.2.1.-, a polypeptide having 6-oxohexanoate dehydrogenase activityclassified under EC 1.2.1.63, a polypeptide having 5-oxopentanoatedehydrogenase activity classified under EC 1.2.1.20, a polypeptidehaving aldehyde dehydrogenase activity classified under EC 1.2.1.3, or apolypeptide having cytochrome P450 monooxygenase activity classifiedunder EC 1.14.14.-.
 11. The method of claim 10, said method furthercomprising enzymatically converting glutarate semialdehyde to5-aminopentanoate using a polypeptide having ω-transaminase activityclassified under EC 2.6.1.-, the method optionally further comprisingenzymatically converting 5-aminopentanoate to cadaverine.
 12. The methodof claim 10, said method further comprising enzymatically convertingglutarate semialdehyde to cadaverine.
 13. The method of claim 11,wherein 5-aminopentanoate is enzymatically converted to cadaverine usinga polypeptide having carboxylate reductase activity classified under EC1.2.99.6 and/or a polypeptide having ω-transaminase activity classifiedunder EC 2.6.1.-, and optionally a polypeptide having N-acetyltransferase activity classified under EC 2.3.1.32 and/or a polypeptidehaving acetylputrescine deacetylase activity classified under EC3.5.1.17 or EC 3.5.1.62.
 14. The method of claim 13, wherein thepolypeptide having carboxylate reductase is enhanced by the gene productof sfp from Bacillus subtilis or the gene product of npt from Nocardia.15. The method of claim 5, wherein pentanoyl-CoA is produced fromacetyl-CoA and propanoyl-CoA using (i) a polypeptide havingβ-ketothiolase activity classified under EC 2.3.1.16 or EC 2.3.1.174 ora polypeptide having acetyl-CoA carboxylase activity classified under EC6.4.1.2. and a polypeptide having β-ketoacyl-[acp] synthase activityclassified under EC 2.3.1.41, EC 2.3.1.179, or EC 2.3.1.180, (ii) apolypeptide having 3-hydroxyacyl-CoA dehydrogenase activity classifiedunder EC 1.1.1.35, EC 1.1.1.36, or EC 1.1.1.157 or a polypeptide having3-oxoacyl-CoA reductase activity classified under EC 1.1.1.100, (iv) apolypeptide having enoyl-CoA hydratase activity classified under EC4.2.1.17 or EC 4.2.1.119, and (v) a polypeptide having trans-2-enoyl-CoAreductase activity classified under EC 1.3.1.8, EC 1.3.1.38, or EC1.3.1.44 to form pentanoyl-CoA from acetyl-CoA and propanoyl-CoA.
 16. Amethod of enzymatically producing 5-hydroxypentanoate, the methodcomprising: a) enzymatically converting pentanoate to pentanoate methylester using a polypeptide having fatty acid O-methyltransferase activityclassified under EC 2.1.1.15; b) enzymatically converting heptanoatemethyl ester to 5-hydroxypentanoate methyl ester using a polypeptidehaving monooxygenase activity classified under EC 1.14.14.- or EC1.14.15.-; and c) enzymatically converting 5-hydroxypentanoate methylester to 5-hydroxypentanoate using a polypeptide having demethylaseactivity classified under EC 2.1.1.- or a polypeptide having esteraseactivity classified under EC 3.1.1.-.
 17. A method of enzymaticallyproducing 5-hydroxypentanoate, the method comprising: a) enzymaticallyconverting pentanoyl-CoA to pentanoic acid pentyl ester using apolypeptide having alcohol O-acetyltransferase activity classified underEC 2.3.1.84; b) enzymatically converting pentanoic acid pentyl ester to5-hydroxypentanoic acid pentyl ester, pentanoic acid 5-hydroxypentylester, or 5-hydroxypentanoic acid 5-hydroxypentyl ester using apolypeptide having monooxygenase activity classified under EC 1.14.14.-or EC 1.14.15.-; and c) enzymatically converting 5-hydroxypentanoic acidpentyl ester, pentanoic acid 5-hydroxypentyl ester, or5-hydroxypentanoic acid 5-hydroxypentyl ester to 5-hydroxypentanoateusing a polypeptide having esterase activity classified under EC3.1.1.-.
 18. The method of claim 1, wherein said recombinant host issubjected to a cultivation strategy under aerobic, anaerobic, ormicro-aerobic cultivation conditions.
 19. The method of claim 18,wherein said recombinant host is cultured under conditions of nutrientlimitation.
 20. The method of claim 18, wherein said recombinant host isretained using a ceramic membrane to maintain a high cell density duringfermentation.
 21. The method of claim 18, wherein the principal carbonsource fed to the recombinant host derives from a biological feedstock.22. The method of claim 21, wherein the biological feedstock is, orderives from, monosaccharides, disaccharides, lignocellulose,hemicellulose, cellulose, lignin, levulinic acid, formic acid,triglycerides, glycerol, fatty acids, agricultural waste, condenseddistillers' solubles, or municipal waste.
 23. The method of claim 18,wherein the principal carbon source fed to the recombinant host derivesfrom a non-biological feedstock.
 24. The method of claim 23, wherein thenon-biological feedstock is, or derives from, natural gas, syngas,CO₂/H₂, methanol, ethanol, benzoate, non-volatile residue (NVR) causticwash waste stream from cyclohexane oxidation processes, or terephthalicacid/isophthalic acid mixture waste streams.
 25. The method of claim 18,wherein the recombinant host is a prokaryote selected from Escherichia,Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Delftia, Bacillus,Lactobacillus, Lactococcus, and Rhodococcus, or a eukaryote selectedfrom Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia,Debaryomyces, Arxula, and Kluyveromyces.
 26. A recombinant hostcomprising at least one exogenous nucleic acid encoding (i) apolypeptide having fatty acid O-methyltransferase activity and (ii) apolypeptide having monooxygenase activity, said recombinant hostproducing a terminal hydroxyl (C₃₋₈ hydroxyalkyl)-C(═O)OCH₃ ester,wherein said polypeptide having fatty acid O-methyltransferase activityis classified under EC 2.1.1.15 and has at least 85% sequence identityto an amino acid sequence set forth in SEQ ID NO:23, SEQ ID NO:24, orSEQ ID NO:25 and said polypeptide having monooxygenase activity isclassified under EC 1.14.14.- or EC 1.14.15.- and has at least 85%sequence identity to an amino acid sequence set forth in SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:28, or SEQ ID NO:29, therecombinant host optionally further comprising an exogenous polypeptidehaving demethylase activity classified under EC 2.1.1.- or a polypeptidehaving esterase activity classified under EC 3.1.1.- and has at least85% sequence identity to the amino acid sequence set forth in SEQ ID NO:27, said host further producing a terminal hydroxyl C₄₋₉hydroxyalkanoate.
 27. The recombinant host of claim 26, wherein saidterminal hydroxyl (C₃₋₈ hydroxyalkyl)-C(═O)OCH₃ ester is5-hydroxypentanoate methyl ester, and is enzymatically converted to5-hydroxypentanoate using an exogenous polypeptide having demethylaseactivity classified under EC 2.1.1.- or a polypeptide having esteraseactivity classified under EC 3.1.1.-, wherein said polypeptide havingesterase activity has at least 85% sequence identity to the amino acidsequence set forth in SEQ ID NO: 27, the recombinant host optionallyfurther comprising one or more exogenous polypeptides selected from apolypeptide having cytochrome P450 monooxygenase activity classifiedunder EC 1.14.14.-; a polypeptide having alcohol dehydrogenase activityclassified under EC 1.1.1.-, a polypeptide having aldehyde dehydrogenaseactivity classified under EC 1.2.1.3, a polypeptide having6-hydroxyhexanoate dehydrogenase activity classified under EC 1.1.1.258,a polypeptide having 6-oxohexanoate dehydrogenase activity classifiedunder EC 1.2.1.63, and a polypeptide having 5-oxopentanoatedehydrogenase activity classified under EC 1.2.1.-, said recombinanthost optionally further producing glutarate semialdehyde or glutaricacid.
 28. The recombinant host of claim 27, said recombinant hostfurther comprising an exogenous polypeptide having ω-transaminaseactivity classified under EC 2.6.1.-, said recombinant host furtherproducing 5-aminopentanoate, the recombinant host optionally furthercomprising an exogenous polypeptide having carboxylate reductaseactivity classified under EC 1.2.99.6 and an exogenous polypeptidehaving ω-transaminase activity classified under EC 2.6.1.- andoptionally one or both of a polypeptide having N-acetyl transferaseactivity classified under EC 2.3.1.32 and a polypeptide havingacetylputrescine deacetylase activity classified under EC 3.5.1.17 or EC3.5.1.62, said host further producing cadaverine.
 29. The recombinanthost of claim 27, said recombinant host further comprising an exogenouspolypeptide having carboxylate reductase activity classified under EC1.2.99.6 and an exogenous polypeptide having alcohol dehydrogenaseactivity classified under EC 1.1.1.-, said host further producing1,5-pentanediol.
 30. The recombinant host of claim 27, said recombinanthost further comprising (i) an exogenous polypeptide havingβ-ketothiolase activity classified under EC 2.3.1.16 or EC 2.3.1.174 oran exogenous polypeptide having acetyl-CoA carboxylase activityclassified under EC 6.4.1.2 and an exogenous polypeptide havingβ-ketoacyl-[acp] synthase activity classified under EC 2.3.1.41, EC2.3.1.179, or EC 2.3.1.180, (ii) an exogenous polypeptide having3-hydroxyacyl-CoA dehydrogenase activity classified under EC 1.1.1.35,EC 1.1.1.36, or EC 1.1.1.157 or an exogenous polypeptide having3-oxoacyl-CoA reductase activity classified under EC 1.1.1.100, (iii) anexogenous polypeptide having enoyl-CoA hydratase activity classifiedunder EC 4.2.1.17 or EC 4.2.1.119, and (iv) an exogenous polypeptidehaving trans-2-enoyl-CoA reductase activity classified under EC 1.3.1.8,EC 1.3.1.38, or EC 1.3.1.44, the recombinant host optionally furthercomprising (v) an exogenous polypeptide having thioesterase activityclassified under EC 3.1.2.- or (vi) an exogenous polypeptide havingbutanal dehydrogenase activity classified under EC 1.2.1.10 or EC1.2.1.57 and an exogenous polypeptide having aldehyde dehydrogenaseactivity classified under EC 1.2.1.3 or EC 1.2.1.4.
 31. A recombinanthost comprising at least one exogenous nucleic acid encoding (i) apolypeptide having alcohol O-acetyltransferase activity and (ii) apolypeptide having monooxygenase activity, and producing any of a(C₄₋₉alkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester, a (C₄₋₉hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester, or a (C₄₋₉hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester, wherein said polypeptide havingalcohol O-acetyltransferase activity is classified under EC 2.3.1.84 andhas at least 85% sequence identity to the amino acid sequence set forthin SEQ ID NO: 26 and said polypeptide having monooxygenase activity isclassified under EC 1.14.14.- or EC 1.14.15.- and has at least 85%sequence identity to an amino acid sequence set forth in SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:28, or SEQ ID NO:29, whereinsaid (C₄₋₉alkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl) ester is 5-hydroxypentanoicacid pentyl ester, said (C₄₋₉ hydroxyalkyl)-OC(═O)—(C₃₋₈ hydroxyalkyl)ester is 5-hydroxypentanoic acid 5-hydroxypentyl ester, and said (C₄₋₉hydroxyalkyl)-OC(═O)—(C₃₋₈ alkyl) ester is pentanoic acid5-hydroxypentyl ester, the recombinant host optionally furthercomprising an exogenous polypeptide having esterase activity and furtherenzymatically converting pentanoic acid 5-hydroxypentyl ester,5-hydroxypentanoic acid 5-hydroxypentyl ester, or 5-hydroxypentanoicacid pentyl ester to 5-hydroxypentanoate and/or 1,5-pentanediol, whereinsaid polypeptide having esterase activity is classified under EC 3.1.1.-and has at least 85% sequence identity to an amino acid sequence setforth in SEQ ID NO:
 27. 32. The recombinant host of claim 31, saidrecombinant host further comprising one or more exogenous polypeptidesselected from a polypeptide having monooxygenase activity classifiedunder EC 1.14.14.- or EC 1.14.15.-, a polypeptide having alcoholdehydrogenase activity classified under EC 1.1.1.-, a polypeptide havingaldehyde dehydrogenase activity classified under EC 1.2.1.3 or EC1.2.1.4, a polypeptide having 6-hydroxyhexanoate dehydrogenase activityclassified under EC 1.1.1.258, a polypeptide having 5-oxopentanoatedehydrogenase activity classified under EC 1.2.1.20, a polypeptidehaving 6-oxohexanoate dehydrogenase activity classified under EC1.2.1.63, and a polypeptide having 7-oxoheptanoate dehydrogenaseactivity classified under EC 1.2.1.-, said host further producingglutarate semialdehyde or glutaric acid, the recombinant host optionallyfurther comprising an exogenous polypeptide having ω-transaminaseactivity classified under EC 2.6.1.-, said host optionally furtherproducing 5-aminopentanoate.
 33. The recombinant host of claim 32, saidrecombinant host further comprising an exogenous polypeptide havingcarboxylate reductase activity and an exogenous polypeptide havingω-transaminase activity and optionally one or more exogenouspolypeptides selected from a polypeptide having N-acetyl transferaseactivity classified under EC 2.3.1.32, a polypeptide havingacetylputrescine deacetylase activity classified under EC 3.5.1.17 or EC3.5.1.62, and a polypeptide having alcohol dehydrogenase activityclassified under EC 1.1.1.-, said host further producing cadaverine. 34.The recombinant host of claim 31, said recombinant host furthercomprising: one or more exogenous polypeptides selected from: (i) apolypeptide having β-ketothiolase activity classified under EC 2.3.1.16or EC 2.3.1.174 or a polypeptide having acetyl-CoA carboxylase activityclassified under EC 6.4.1.2 and a polypeptide having β-ketoacyl-[acp]synthase activity classified under EC 2.3.1.41, EC 2.3.1.179, or EC2.3.1.180, (ii) a polypeptide having 3-hydroxyacyl-CoA dehydrogenaseactivity classified under EC 1.1.1.35, EC 1.1.1.36, or EC 1.1.1.157 or apolypeptide having 3-oxoacyl-CoA reductase activity classified under EC1.1.1.100, (iii) a polypeptide having enoyl-CoA hydratase activityclassified under EC 4.2.1.17 or EC 4.2.1.119, and (iv) a polypeptidehaving trans-2-enoyl-CoA reductase activity classified under EC 1.3.1.8,EC 1.3.1.38, or EC 1.3.1.44; or one or more exogenous polypeptidesselected from a polypeptide having carboxylate reductase activityclassified under EC 1.2.99.6, a polypeptide having aldehydedehydrogenase activity classified under EC 1.2.1.3 or EC 1.2.1.4, apolypeptide having butanal dehydrogenase activity classified under EC1.2.1.10 or EC 1.2.1.57, and a polypeptide having alcohol dehydrogenaseactivity classified under EC 1.1.1.-.
 35. The method of claim 8, whereinthe method further comprises enzymatically converting 5-hydroxypentanoicacid 5-hydroxypentyl ester or 5-hydroxypentanoic acid pentyl ester to5-hydroxypentanoate, said enzymatic conversion of 5-hydroxypentanoicacid 5-hydroxypentyl ester or 5-hydroxypentanoic acid pentyl ester to5-hydroxypentanoate using a polypeptide having esterase activityclassified under EC 3.1.1.-, said method further comprisingenzymatically converting 5-hydroxypentanoate to cadaverine or1,5-pentanediol.
 36. The method of claim 35, wherein 5-hydroxypentanoateis enzymatically converted to cadaverine using a polypeptide havingcarboxylate reductase activity classified under EC 1.2.99.6, apolypeptide having ω-transaminase activity classified under EC 2.6.1.-,and a polypeptide having alcohol dehydrogenase activity classified underEC 1.1.1.-.
 37. The method of claim 35, wherein 5-hydroxypentanoate isenzymatically converted to 1,5-pentanediol a polypeptide havingcarboxylate reductase activity classified under EC 1.2.99.6 and apolypeptide having alcohol dehydrogenase activity classified under EC1.1.1.-, the method optionally further comprising enzymaticallyconverting 1,5-pentanediol to cadaverine using a polypeptide havingalcohol dehydrogenase activity classified under EC 1.1.1.- and apolypeptide having ω-transaminase activity classified under EC 2.6.1.-.38. The method of claim 11, wherein said polypeptide havingω-transaminase activity classified under EC 2.6.1.- has at least 85%sequence identity to any one of the amino acid sequences set forth inSEQ ID NO. 8-13.
 39. The method of claim 19, wherein said recombinanthost is cultured under conditions of phosphate, nitrogen, or oxygenlimitation.
 40. The method of claim 4, said method further comprisingenzymatically converting 5-hydroxypentanoate methyl ester to5-hydroxypentanoate using a polypeptide having demethylase activityclassified under EC 2.1.1.- or a polypeptide having esterase activityclassified under EC 3.1.1.-.
 41. The method of claim 40, said methodfurther comprising enzymatically converting 5-hydroxypentanoate toglutarate semialdehyde, said enzymatic conversion of 5-hydroxypentanoateto glutarate semialdehyde using: a polypeptide having alcoholdehydrogenase activity classified under EC 1.1.1.-, a polypeptide having6-hydroxyhexanoate dehydrogenase activity classified under EC 1.1.1.258,or a polypeptide having cytochrome P450 monooxygenase activityclassified under EC 1.14.14.-, the method optionally further comprisingenzymatically converting glutarate semialdehyde to glutaric acid using apolypeptide having 7-oxoheptanoate dehydrogenase activity classifiedunder EC 1.2.1.-, a polypeptide having 6-oxohexanoate dehydrogenaseactivity classified under EC 1.2.1.63, a polypeptide having5-oxopentanoate dehydrogenase activity classified under EC 1.2.1.20, apolypeptide having aldehyde dehydrogenase activity classified under EC1.2.1.3, or a polypeptide having cytochrome P450 monooxygenase activityclassified under EC 1.14.14.-.
 42. The method of claim 41, said methodfurther comprising enzymatically converting glutarate semialdehyde to5-aminopentanoate using a polypeptide having ω-transaminase activityclassified under EC 2.6.1.-, the method optionally further comprisingenzymatically converting 5-aminopentanoate to cadaverine.
 43. The methodof claim 41, said method further comprising enzymatically convertingglutarate semialdehyde to cadaverine.
 44. The method of claim 42,wherein 5-aminopentanoate is enzymatically converted to cadaverine usinga polypeptide having carboxylate reductase activity classified under EC1.2.99.6 and/or a polypeptide having ω-transaminase activity classifiedunder EC 2.6.1.-, and optionally a polypeptide having N-acetyltransferase activity classified under EC 2.3.1.32 and/or a polypeptidehaving acetylputrescine deacetylase activity classified under EC3.5.1.17 or EC 3.5.1.62.
 45. The method of claim 44, wherein thepolypeptide having carboxylate reductase is enhanced by the gene productof sfp from Bacillus subtilis or the gene product of npt from Nocardia.46. The method of claim 40, said method further comprising enzymaticallyconverting 5-hydroxypentanoate to cadaverine or 1,5-pentanediol.
 47. Themethod of claim 46, wherein 5-hydroxypentanoate is enzymaticallyconverted to 1,5-pentanediol a polypeptide having carboxylate reductaseactivity classified under EC 1.2.99.6 and a polypeptide having alcoholdehydrogenase activity classified under EC 1.1.1.-, the methodoptionally further comprising enzymatically converting 1,5-pentanediolto cadaverine using a polypeptide having alcohol dehydrogenase activityclassified under EC 1.1.1.- and a polypeptide having ω-transaminaseactivity classified under EC 2.6.1.-.